Electrochemical properties of S-Adenosyl-L-Methionine and studies of a chimeric radical Sam enzyme-flavodoxin fusion protein

Members of the Radical SAM (RS) enzyme superfamily utilize the reductive capability of a coordinated [4Fe-4S] cluster to generate a chemically useful C-centered radical arising from the scission of S-adenosyl-L-methionine (SAM). In this dissertation, a dual approach towards following electron movements into both the [4Fe-4S] cluster and SAM itself were utilized. Characterization of the electrochemical properties of SAM in bulk solution showed homolysis occurs at a more permissive cathodic peak potential (Epc) -1.4V compared to previous literature estimates. Additionally, the biochemical and kinetic effects arising from the fusion of an activation domain to a catalytically competent domain were determined. Together these results have furthered the field by establishing an upper limit to the energetic requirement for the homolysis of SAM in bulk solution and the effects on [4Fe-4S] cluster +2/+1 reduction arising from activator proximity.

ΕLECTROCHEMICAL PROPERTIES OF S-ADENOSYL-L-METHIONINE AND STUDIES OF A CHIMERIC RADICAL SAM ENZYME-FLAVODOXIN FUSION PROTEIN by Sven Alexander Miller A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry The University of Utah December 2019 Copyright © Sven Alexander Miller 2019 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL Sven Alexander Miller The dissertation of has been approved by the following supervisory committee members: Vahe Bandarian , Chair 8/14/2019 Date Approved Peter Francis Flynn , Member 8/14/2019 Date Approved Matthew T. Kieber-Emmons , Member 8/14/2019 Date Approved Ryan E. Looper , Member 8/14/2019 Date Approved Martin P. Horvath , Member 8/14/2019 Date Approved and by Vahe Bandarian the Department/College/School of and by David B. Kieda, Dean of The Graduate School. , Chair/Dean of Chemistry ABSTRACT Members of the Radical SAM (RS) enzyme superfamily utilize the reductive capability of a coordinated [4Fe-4S] cluster to generate a chemically useful C-centered radical arising from the scission of S-adenosyl-L-methionine (SAM). In this dissertation, a dual approach towards following electron movements into both the [4Fe-4S] cluster and SAM itself were utilized. Characterization of the electrochemical properties of SAM in bulk solution showed homolysis occurs at a more permissive cathodic peak potential (Epc) -1.4V compared to previous literature estimates. Additionally, the biochemical and kinetic effects arising from the fusion of an activation domain to a catalytically competent domain were determined. Together these results have furthered the field by establishing an upper limit to the energetic requirement for the homolysis of SAM in bulk solution and the effects on [4Fe-4S] cluster +2/+1 reduction arising from activator proximity. TABLE OF CONTENTS ABSTRACT ...................................................................................................................... iii LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii LIST OF ABBREVIATIONS ............................................................................................ ix Chapters 1. S-ADENOSYL-L-METHIONINE AND RADICAL ENZYME CHEMISTRY ........... 1 1.1 Introduction .......................................................................................................... 2 1.2 Benchmark Findings in Sulfonium Salt Electrochemistry................................... 2 1.3 Biological Relevance of Sulfonium Salts ............................................................ 3 1.4 Biosynthesis of SAM ........................................................................................... 3 1.5 SAM as an Initiator of Radical Chemistry........................................................... 6 1.6 Classification of Radical SAM Enzymes Based on SAM Utilization ................. 9 1.7 Structural Features Direct Radical Fate ............................................................. 10 1.8 SAM as a Source of Methyl, 2-Amino-2-Carboxypropyl, or dAdo Groups in Radical SAM Enzymes ............................................................................................ 13 1.9 Reduction Potential of [4Fe-4S] Clusters .......................................................... 14 1.9.1 Biological and Chemical Reduction of [4Fe-4S] Clusters..................... 14 1.9.2 Redox Potential of the [4Fe-4S] Cluster Is Altered by Substrate/Cofactor Binding............................................................................................................ 15 1.10 Models of Homolysis of SAM ......................................................................... 17 1.10.1 Direct Electron Transfer ...................................................................... 17 1.10.2 Ω Intermediate ..................................................................................... 19 1.11 Scope of Dissertation ....................................................................................... 19 1.12 References ........................................................................................................ 21 2. ANALYSIS OF THE ELECTROCHEMICAL PROPERTIES OF S-ADENOSYL-LMETHIONINE AND IMPLICATIONS FOR ITS ROLE IN RADICAL SAM ENZYMES........................................................................................................................ 29 2.1 Abstract .............................................................................................................. 30 2.2 Introduction ........................................................................................................ 30 2.3 Results and Discussion ...................................................................................... 32 2.3.1 Cyclic Voltammetry of Bulk SAM ........................................................ 32 2.3.2 SAM Is Cleaved Under Reducing Conditions to Produce dAdo ........... 40 2.3.3 dAdo Forms in a One Electron Process ................................................. 43 2.3.4 Reductive Cleave of SAM also Produces 2-Aminobutyrate ................. 47 2.4 Conclusions ........................................................................................................ 53 2.5 Material and Methods ........................................................................................ 53 2.5.1 Enzymatic Preparation of SAM ............................................................. 53 2.5.2 Cyclic Voltammetry of SAM ................................................................. 53 2.5.3 Controlled Potential of Electrolysis of SAM ......................................... 54 2.5.4 Analysis of Products of Controlled Potential Electrolysis of SAM....... 55 2.5.5 Analysis of Homolysis of 5'-deoxyadenosylcobalamin (AdoCbl) ........ 55 2.6 References .......................................................................................................... 56 3. MECHANISM OF ACTIVATION FOR A CHIMERIC RADICAL SAM ENZYMEFLAVODOXIN FUSION PROTEIN ............................................................................... 58 3.1 Abstract .............................................................................................................. 59 3.2 Introduction ........................................................................................................ 59 3.3 Results and Discussion ...................................................................................... 65 3.3.1 Purification and Cofactor Analysis ........................................................ 65 3.3.2 ECF Is Catalytically Active ................................................................... 66 3.3.3 Is Activation of ECF Facilitated by Intermolecular or Intramolecular Reduction? ...................................................................................................... 72 3.4 Conclusions ........................................................................................................ 78 3.5 Materials and Methods....................................................................................... 78 3.5.1 Protein Expression and Purification....................................................... 78 3.5.1.1 E. coli QueE-FldA Fusion ........................................................... 78 3.5.1.2 E. coli QueE ............................................................................... 81 3.5.1.3 E. coli Flavodoxin ....................................................................... 81 3.5.2 Amino Acid Analysis............................................................................. 81 3.5.3 Elemental Iron Content .......................................................................... 82 3.5.4 Sulfide Content ...................................................................................... 82 3.5.5 FMN Content ......................................................................................... 82 3.5.6 Synthesis of Substrates .......................................................................... 83 3.5.7 Steady-State Kinetic Analysis of QueE ................................................. 83 3.5.8 Spectrophotometric Reduction with Dithionite ..................................... 83 3.6 References .......................................................................................................... 84 4. SUMMARY OF DISSERTATION .............................................................................. 88 4.1 Summary Statement ........................................................................................... 89 v LIST OF TABLES Tables 1.1 Summary of Redox Potentials for Selected Proteins .................................................. 16 3.1 Cofactor Quantification of Purified Proteins .............................................................. 69 LIST OF FIGURES Figures 1.1. Biological Schemes for the Production of SAM.......................................................... 5 1.2. Redox Chemistry of [4Fe-4S] Cluster and Utilization of SAM. ................................. 8 1.3. Structural Features of RS Enzymes. .......................................................................... 11 1.4. [4Fe-4S] and SAM Redox Potentials Throughout the Catalytic Cycle of LAM. ...... 18 1.5. SAM Homolysis Models. .......................................................................................... 20 2.1. Cyclic Voltammograms of SAM. .............................................................................. 33 2.2. Dependence of SAM Reductive Peak Current on Scan Rate .................................... 35 2.3. Cyclic Voltammograms of Methyl Viologen (MV). ................................................. 36 2.4. Solvent and Scan Rate Dependence of Reductive Wave and Currents in CV Experiments with SAM..................................................................................................... 37 2.5. Cyclic Voltammetry of Commerical (gray) and Enzymatically Prepared SAM (black). .............................................................................................................................. 38 2.6. Glassy-Carbon Working Electrode Functionalization. .............................................. 39 2.7. SAM Is Cleaved Under Reducing Conditions to Produce dAdo. .............................. 41 2.8 Isotope Incorporation from Bulk Solvent into dAdo. ................................................. 42 2.9. Analysis of Photolysis Products of AdoCbl. ............................................................. 44 2.10. Fragmentation of Authentic cyc-dAdo Standard in the HCD Cell at Various Power Settings. ................................................................................................................. 46 2.11. Characterization of m/z 250 Species from Controlled Potential Electrolysis of SAM. ................................................................................................................................. 48 2.12. EIC Trace at m/z 250 for a cyc-dAdo Standard. ...................................................... 49 2.13. Controlled Potential Electrolysis of SAM Produces 2-AB. ..................................... 50 2.14. dAdo and 2-AB Are Produced with Similar Rates During Controlled Potential Electrolysis of SAM.......................................................................................................... 52 3.1. Biosynthesis Pathway to 7-deazapurine tRNA Base Queuosine. .............................. 61 3.2. Radical Mediated Ring Contraction Mechanism of QueE. ....................................... 62 3.3. Characterization of Protein Purity. ............................................................................ 67 3.4. UV-Visible Overlay of Purified Proteins................................................................... 68 3.5. Chemical and Biological Reduction of ECF. ............................................................. 71 3.6. Progress Curves for Formation of CDG. ................................................................... 73 3.7. Spectroscopic Reduction of FldA with DT................................................................ 75 3.8. Spectroscopic Reduction of FldA with DT in the Presence of MV. .......................... 76 3.9. Spectroscopic Reduction Replot of Oxidized and Semiquinone Features................. 77 3.10. Protein Sequence of E. coli Chimeric Fusion Protein.............................................. 79 viii LIST OF ABBREVIATIONS 2-AB Ade ATP AdoCbl BioB CPH4 QueE CDG C-H Epc HemN CV Cyc-dAdo dAdo dAdo• DTB D D2 O e Dph2 DT EPR EF2 E. coli EXAFS ΕIC Fd Fld FldA GD HCD HPLC H HydG L-Met LipA LC-MS LAM m/z 2-aminobutyrate Adenine Adenosine triphosphate Adenosylcobalamin Biotin synthase 6-carboxy-5,6,7,8-tetrahydropteirn 7-carboxy-7-deazaguanine synthase 7-carboxydeazaguanine Carbon-hydrogen bond Cathodic peak potential Coproporphyrinogen III oxidase Cyclic voltammetry 8,5’-cyclodeoxyadenosine 5’-deoxyadenosine 5’-deoxyadenosyl radical Desthiobiotin Deuterium Deuterium oxide Dielectric constants Diphthamide biosynthesis protein 2 Dithionite Electron paramagnetic resonance Elongation factor-2 Escherichia coli Extended X-ray Absorption Fine Structure Extracted ion chromatogram Ferrodoxins Flavodoxin Flavodoxin A Glycerol dehydratase High-energy collisional dissociation High pressure liquid chromatography Hydrogen Hydrogenase maturase L-methionine Lipoic acid synthase Liquid chromatography-mass spectrometry Lysine 2,3-aminomutase Mass to charge ratio v MV ppm ip ThiC KBr KCl KI PFL PFL-AE RS SAM SCE SHE TIM Methyl Viologen Parts per million Peak current Phosphomethylpyrimidine synthase Potassium bromide Potassium chloride Potassium iodide Pyruvate Formate-Lyase Pyruvate Formate-Lyase Activating Enzyme Radical SAM S-adenosyl-L-methionine Standard calomel electrode Standard hydrogen electrode Triose phosphate isomerase x CHAPTER 1 S-ADENOSYL-L-METHIONINE AND RADICAL ENZYME CHEMISTRY 2 1.1. Introduction The commonly attributed ability of sulfonium salts to produce a C-centered radical over a wide range of reduction potentials is leading to an increased interest in leveraging their reactivity towards industrial applications.1-2 There is also growing interest in understanding their role in a wide range of biochemical processes.3 The first part of this chapter will review the physical properties of sulfonium salts in solution. The second half will focus on their biochemical utility through specific focus on an expansive family of enzymes that perform radical-mediated transformations. 1.2. Benchmark Findings in Sulfonium Salt Electrochemistry Synthesis of di- and tri-substituted sulfur centers using alkyl bromides 4 allows for the rapid generation of compound libraries to perform comparative studies of the redox properties of the C-S bonds. Initial polarographic studies on the electrochemical properties of the cyclic thioether linkages in p-rhodaniline 5 or the linear thioether S-alkythiouronium salt 6 were expanded by Colichman et al. to tri-substituted sulfur compounds in 1953.7 In this study, reduction of the trimethylsulfonium salt produced a single wave peak at -1.82 V against the standard calomel electrode (SCE). Since polarographic experiments only progress in the cathodic direction, oxidative features in the anodic direction cannot be observed, and a threshold of slope steepness must be met to indicate the reductive wave generated arises from a nonreversible process. Slope fitting analysis of the trimethylsulfonium salt wave (0.113) was found to be considerably above the upper limit for a reversible chemical process (0.059), suggesting that the measured peak potential of 1.82 V represents an upper limit on the reduction potential of the trimethylsulfonium salt. 3 Nevertheless, the value determined in this study has served as a benchmark for the biologically relevant sulfonium S-adenosyl-L-methionine (SAM), which is a cofactor in many complex radical-mediated transformations (reviewed in Broderick et al. 2014).8 However, prior to the work presented in this dissertation, there has been no studies of the electrochemical properties of SAM. 1.3. Biological Relevance of Sulfonium Salts Pioneering studies conducted in the 1930s focused specifically on the physiological role of L-methionine (Met) revealed that neither homocysteine nor homocystine could replace this amino acid as a dietary supplement to sustain growth in rat models unless additional compounds were also present.9 A decade later, Tonnies and colleagues described the total synthesis and biochemical utility 10-12 of numerous sulfonium compounds derived from L-Met. However, it was not until 1948 when isolation of a sulfonium salt from marine alga,13 attributed to the conversion of this amino acid to cysteine,14 did transmethylation in nature become the topic of numerous investigations that remain active areas of research currently. 1.4. Biosynthesis of SAM Initially, the link between L-Met and its biological activity remained elusive. However, once the intimate involvement of adenosine triphosphate (ATP) in the adenylation of L-Met was discovered, the mechanism for conversion of the amino acid into its biologically active form become apparent.15 Initial production of “active methionine” required the combination of ATP, L-Met, and liver extracts to form what is 4 now known as S-adenosyl-L-methionine (SAM) (Figure 1.1A). Pulse-chase experiments with 3H-labeled L-Met led to rapid incorporation into SAM, with half maximal time of 30 sec.16 The basal levels of SAM in yeast are ~0.3-0.8 µmol per g of wet cells. However, exogenous addition of Met to media can raise intracellular SAM levels 50- to 100-fold. Conversely, supplementing media with SAM inhibits its production.17 SAM has two chiral centers (α-carbon and sulfonium), and therefore it can have four possible diastereomers. Experimentally it has been found that only one of the two possible isomers of the sulfonium are produced from the biological systems studied. The critical enzyme isolated from liver extracts, which catalyzes the production of SAM in vitro, was initially referred to as methionine-activating enzyme but was later coined SAM synthetase.10-12 Early characterization of SAM synthetase showed the complete dephosphorylation of ATP, which was unique for the times. High selectivity for ATP as a substrate was also demonstrated,18 since even closely related inosine triphosphate and 2’-deoxyadenosine triphosphate were not converted into SAM. Furthermore, other structurally related nucleotides analogs competitively inhibit SAM production. These early observations highlight that the triphosphate moiety of ATP and enzyme specificity for the ribose and adenine are significant determinants of chemistry and recognition.18 By contrast, SAM synthetase will incorporate α-hydroxy compound 2-hydroxy-4-methylthio-n-butyric acid and the S-ethyl analogs of L-Met into the final products, albeit at slower rates.18 The biosynthesis of SAM from ATP and L-Met by SAM synthetase (Figure 1.1A), initially characterized by Mudd and Mann,18 occurs in an overall four step mechanism. Upon binding of ATP and subsequent recruitment of L-Met to the enzyme active site 5 Figure 1.1 – Biological Schemes for the Production of SAM. A) Structures of the major components required to produce “active methionine”, i.e., SAM, B) The kinetic mechanism of SAM synthetase involves binding of ATP and Met, followed by formation of SAM. Cleavage of triphosphate to diphosphate and phosphate is a major driving force in the transformation. 6 (Figure 1.1B), the rate limiting adenosyl transfer step causes triphosphate cleavage and formation of SAM (Figure 1.1B step iii). Subsequent enzyme-catalyzed decomposition of the triphosphate to pyrophosphate and inorganic phosphate prevents the reverse reaction from proceeding. Enhanced binding affinities of SAM and triphosphate compared to those of ATP, L-Met, diphosphate, and phosphate further help drive the overall equilibrium of this reaction toward product formation. While SAM synthetase does undergo product inhibition, low levels of SAM have been shown to stimulate enzymatic activity.18-19 However, when the demand for this cofactor is high or production is stimulated, harmful drops in ATP levels have been observed, but may be mitigated with exogenous addition of adenine.18 As a whole, numerous feedback loops exist to regulate SAM synthesis and its effects on cellular homeostasis, which highlights the central role of this cofactor in the cell. 1.5. SAM as an Initiator of Radical Chemistry While the most widely recognized function for SAM is methyl transfer, pioneering studies by Knappe, Frey, and colleagues in the 1980s demonstrated that SAM is the key cofactor in reactions that involve radical mediated transformations.20-22 The class of enzymes that harness the sulfonium moiety to carry out radical-mediated transformations at unactivated carbon centers is present in virtually every aspect of physiology across all kingdoms of life.23-24 Bioinformatic analysis using the CX3CX2C protein sequence motif and in addition to operon type, biochemical pathway and domain of life, have since identified over >110,000 distinct members of the now coined radical SAM (RS) superfamily.25-27 Numerous members perform key steps in essential biochemical processes, across all three 7 domains of life, thereby demonstrating the utility of radicals to perform otherwise intractable chemistry in nature. With such a vast array of diversity and only a handful of well characterized RS enzymes, much of this field remains to be explored. The remaining sections of this chapter will delve into the structural and mechanistic details of the enzymes in the RS superfamily. The unifying property of all RS SAM enzymes is the coordination of a [4Fe-4S] site-differentiated cubane cluster, through three cysteine sidechains of the bioinformatically identified conserved CX3CX2C motif 28-29 (reviewed in Broderick et al. 2014).8As isolated, RS enzymes are inactive and require the presence of a reductant for function. In the resting state, the cluster is in a +2 oxidation state. Reduction of the cluster from the +2 to the +1 state has been proposed to be followed by reductive fragmentation of SAM to generate a 5’-deoxyadenosyl radical (dAdo•) and L-Met.20, 30-33 In most radical SAM enzymes, the dAdo• catalyzes hydrogen (H) -atom abstraction to initiate the transformation of the substrate, the details of which are unique to each system. The mechanistic model depicted in Figure 1.2 summarizes key features of the catalytic cycle common to most radical SAM enzymes. Extended X-ray absorption fine structure (EXAFS) spectroscopy measurements of the RS enzymes lysine 2,3aminomutase (LAM), using a selenium analog in place of sulfur in SAM, indicates the Se atom of Met is within 2.7 Å of the iron atom in the cluster.29 Furthermore, SAM interacts directly with the unique iron atom of the cluster through its amino and carboxylate moieties (Figure 1.2). This unique coordination was first observed with pyruvate formate-lyase Activating Enzyme (PFL-AE),28, 34 but it has since been shown to be a common feature of nearly all RS enzymes. Finally, large numbers of structural studies across the superfamily 8 Figure 1.2 – Redox Chemistry of [4Fe-4S] Cluster and Utilization of SAM. Reductive cleavage of SAM generates dAdo•, which initiates radical-mediated transformations. In some RS enzymes, SAM is regenerated at the end of the catalytic cycle, whereas in others, SAM is used stoichiometrically. 9 place the abstracted H atom generally within van der Waal radius of the 5’-position of the cofactor. 35-37 1.6. Classification of Radical SAM Enzymes Based on SAM Utilization Radical SAM enzymes catalyze various chemical reactions in the biosynthesis of vitamins, antibiotics cofactors, DNA and RNA methylation, in addition to protein modifications.38-39 Mechanistically, these enzymes can be categorized into three main classes (I, II, and III) depending on the fate of SAM. The most well studied member of the Class I RS enzymes is LAM, which performs the conversion of L-a-lysine to L-β-lysine 40 (consistent with Figure 1.2-catalytic cycle). During this reaction, SAM is reductively cleaved to dAdo•, which abstracts a substrate Hatom in a regio- and stereoselective manner to generate a substrate radical. Rearrangement of the substrate-like intermediate to a product-like one is followed by abstraction of a Hatom from 5’-deoxyadenosine (dAdo) to form product. In Class I enzymes, dAdo recombines with methionine to regenerate SAM, reducing the [4Fe-4S] cluster to the catalytic +1 state. SAM is used catalytically. LAM remains the best characterized of these enzymes where nearly every postulated intermediate has been observed directly by electron paramagnetic resonance (EPR) spectroscopy.30-31, 41 QueE, which catalyzes the conversion of 6-carboxy-5,6,7,8-tetrahydropteirn (CPH4) to 7-carboxydeazaguanine (CDG), is also a Class I enzyme. It catalyzes the formation of a key intermediate in the biosynthesis of pyrrolpyrimidine containing natural products and nucleic acids. Studies on the reductive activation of QueE will be the topic of Chapter 3. 10 In contrast to Class I enzymes, Class II radical SAM enzymes utilize SAM as a substrate and do not regenerate it at the end of the catalytic cycle (Figure 1.2-substrate SAM). In these enzymes homolysis of SAM serves to generate dAdo•, which subsequently abstracts a H-atom from a peptide glycine to generate a glycyl radical. It is the glycyl radical that subsequently initiates the catalytic cycle by H-atom transfer events. The best studied Class II radical SAM enzyme is pyruvate formate lyase activating enzyme, which generates the essential intermediate glycyl radical on PFL that is necessary for function.28, 34 In these enzymes, a single activated glycyl species could serve to initiate many turnover cycles. Class III enzymes use SAM as a substrate and produce dAdo and Met in stoichiometric amounts per cycle of substrate turnover. The SAM does not re-form at the end of the catalytic cycle. Class III enzymes are exemplified by biotin synthase (BioB) and lipoic acid synthase (LipA), which catalyzes the insertion of sulfur into unactivated carbonhydrogen (C-H) bonds.42-46 1.7. Structural Features Direct Radical Fate Structural studies of the RS superfamily have revealed that the “core fold” structure is either a full (α/β)8 or partial (α/β)6 triose phosphate isomerase (TIM) barrel. The interior parallel ß strands of the barrel are surrounded by α helices that form the protein surface (Figure 1.3A). At the opening of the barrel, the thiolate sidechains of the cysteine residues in the CX3CX2C motif coordinate the [4Fe-4S] cluster which binds and activate SAM.8, 36 Despite low overall sequence similarity between individual RS enzymes, overlay of Ca positions are commonly within 2-4 Å root mean square deviation.8 Diversification of the transformations performed by these enzymes is mainly achieved through utilization of 11 Figure 1.3 – Structural Features of RS Enzymes. A) Structure of BsQueE (PDB: 5TGS) from backside (i) and front (ii) view with interior b-sheets of TIM barrel highlighted in green and corresponding a helices in magenta that form the protein surface. B) Structure of ThiC (PDB: 3EPO) that highlights the interplay of three distinct domains, which is similar to AdoCbl enzymes; N-terminal domain in light gray, the radical SAM domain comprised of a full TIM barrel in green, and a C-terminal Fe-S cluster binding domain in yellow. C) Dynamic loop movements of PFL-AE from its unbound state (PDB: 3C8F, green with orange loop) to its SAM bound state (PDB: 3CB8, cyan with red loop ). 12 different coenzymes, coordination of oligomeric protein domains, and flexibility of loop regions during catalysis.8 Prominent examples of evolutionary importance and structural adaptation are discussed below. The architectural overlap between enzymes that utilize SAM as a redox cofactor and those which perform one-electron reactions using adenosylcobalamin (AdoCbl) is most identifiable in phosphomethylpyrimidine synthase (ThiC) (Figure 1.3B) which is comprised of a genetic duplicate of two full TIM barrel subunits.47-51 ThiC shields its active site from solvent by folding the cluster-binding loop from one subunit over the neighboring catalytic domain,52 similar to the Rossmann-like domain of AdoCbl-dependent enzymes.36 This achieves proper positioning of SAM and the [4Fe-4S] cluster over substrate for optimal control of the generated radical. Interestingly, the positioning of dAdo in ThiC is closely related to its coordination in BioB.36 These early structures provided a tentative evolutionary link between early examples of radical enzyme chemistry and the rapidly expanding class of enzymes that reduce SAM to initiate chemical transformations. Through optimization of flexible loops and alternative protein conformations upon binding of substrate/cofactor, numerous RS enzymes sequester their active sites from solvent to prevent access to or release of reactive species. In the case of PFL-AE, for example, loops B and C serve to close off the top portion of the TIM barrel while allowing the space for substrate and cofactors to diffuse into the protein core through the remaining bottom or lateral sections (Figure 1.3C).53 In BioB the carboxylate moiety of the desthiobiotin (DTB) interacts with two consecutive threonine residues to promote loop closure.54 Coproporphyrinogen III oxidase (HemN) uses a two loop mechanism to close and partially fill the active site during catalysis.55 One loop, referred to as a “tripwire,” 13 undergoes a disordered to ordered transition up binding of the substrate. Combining both molecular coordination of coenzymes and substrate with the inherent flexibility encoded in the protein provides a dual approach to prevent unwanted side reactions arising from the radicals generated during chemical transformations. 1.8. SAM as a Source of Methyl, 2-amino-2-carboxypropyl, or Deoxyadenosine Groups in Radical SAM Enzymes While the reductive cleave of SAM nearly always leads to the formation of dAdo•, alternative outcomes for both SAM and dAdo• have been documented (Figure 1.2). In diphthamide biosynthesis protein 2 (Dph2) cleavage of SAM produces the 2aminobutyrate (2-AB) radical, which undergoes addition to a His residue of elongation factor-2 (EF2) to generate diphthamide.56-57 In cobalamin-independent glycerol dehydratase (GD)-activating enzyme, 2-AB is utilized to form a glycyl radical on GD to support catalysis. 58 In addition, there are examples of systems where instead of H- atom abstraction, dAdo• undergoes radical addition. In the biogenesis of futolosin 59-60 or peptidylnucleoside antibiotics,61 dAdo• adds to the substrate generating a dAdo- containing product. In yet another variation, a subset of radical SAM enzymes neither use the [4Fe-4S] cluster to bind nor reductively cleave SAM.62 Instead, the methyl group from SAM is transferred to additional cofactors, such as cobalamin, which relays this modification to the substrate. These notable exceptions notwithstanding, production of dAdo• is a key intermediate in the majority of chemical transformations performed by this superfamily. 14 1.9. Reduction Potential of [4Fe-4S] Clusters The unifying principle of SAM activation by radical enzymes is the coordination of a [4Fe-4S] site-differentiated cluster, which binds and poises the cofactor for generation of a catalytic radical (reviewed in Broderick et al. 2014).8This section will review the current state of the field in this area.63-64 Electron transfer events initiated by the wide variety of functionally active iron forms, most important for this discussion are the dinuclear iron complexes [2Fe-2S] and [4Fe-4S], span a wide range of reductions potentials from +360 to -765 mV, which are modulated by their immediate chemical environments.63-64 Understanding the inherent reactivity of the [4Fe-4S]+1 in vitro has provided new insight into the control of catalysis imposed by RS enzymes. 1.9.1. Biological and Chemical Reduction of [4Fe-4S] Clusters In its resting state, the radical [4Fe-4S] cluster responsible for the activation of SAM is most often found in its catalytically inactive +2 state, which much be reduced to its +1 state. Most in vitro studies to date have engaged the +1 state of the cluster by the use of a strong reducing agent such as dithionite (DT).65-66 However, due to the highly charged state of DT, occasionally a less charged chemical mediator may be used to access the [4Fe-4S]. Reductive mediation using methyl viologen (MV) is common due to its closely coupled reducing potential relative to the resting +2 state of the cluster. In the cell, flavodoxins (Fld) and ferrodoxins (Fd) are thought to perform the role of reducing these clusters.33, 67-70 With many enzymes, use of an “in vivo” reducing system serves to maximize catalytic activity. In such cases, the “generic” Escherichia coli 15 (E.coli) flavodoxin A (FldA) (encoded by fldA) has historically served as a surrogate for the naturally occurring protein, which in nearly all cases, is not known. While often at least two flavodoxins are encoded in the bacterial genome, knock-out experiments suggest that they may be performing nonoverlapping functions. Electrochemical studies also demonstrate the redox potentials of these proteins are within range for reduction of radical SAM clusters.71-72 Fld binding is proposed to occur on at the face of the TIM barrel that houses the [4Fe-4S] cluster.53-55 One might, however, imagine therefore that the differences in the electrostatics of this surface may, to some extent, dictate the binding affinities. To date, there has only been one systematic study where activation of and RS enzyme by various native Fld homologs have been compared to the “generic” E. coli protein.73 As highresolution structures of homologs of radical SAM enzymes from different sources are solved, it may be possible to begin to understand the factors that drive these interactions. A more detailed discussion of the biological activation of the radical SAM enzyme 7carboxy-7-deazaguanine synthase (QueE) will be presented in Chapter 3. 1.9.2. Redox Potential of the [4Fe-4S] Cluster Is Altered by Substrate/Cofactor Binding While the reductive potential of SAM was not previously determined, estimates based on the electrochemical properties of other sulfonium salts in solution set the benchmark at -1.8V (vs SCE).7 The midpoint potential of the radical SAM cluster in many members of the superfamily have been measured, and they are all in the range of -430 to -550 mV (Table 1.1). Frey and colleagues utilized LAM as a model system to show that the potential of the cluster can be modulated by binding various ligands.74-75 Upon binding of 16 Table 1.1 – Summary of Redox Potentials for Selected Proteins. Protein Class Midpoint (vs S.H.E) Technique Year References TaFd Ferrodoxin -431/ PFE/Bulk 2000 76 -587mV BioB Sulfur Transferase -430mVb Spectroelectrochemistry 2001 77 LAM Mutase -480mV Spectroelectrochemistry 2006 78 BtrN Dehydrogenase -510/ PFE 2015 64 -765mV TsrM Class B RS methylase -550mV PFE 2016 62 RimO Methylthiotransferase -420c/-405mV PFE 2016 79-80 MiaB Methylthiotransferase -450c/-390mV PFE 2016 80 Tte118 Maturase (thioether crosslink) -450c/-500/- PFE 2018 81 Spectrophotometry 2018 82 Spectrophotometry 2018 82 FldA E. coli Flavodoxin 550mV -149†/ -380§mV Fld2 E. coli Flavodoxin -127†/ -385§ mV a PFE refers to protein film voltammetry BioB contains two distinct clusters (one [2Fe-2S] and one [4Fe-4S]) with indistinguishable redox potentials c Indicates the RC in a multi-cluster radical SAM protein Denotes first (†) and second (§) electron transfer events in a two-electron reduction system. b 17 SAM, the reduction potential of the cluster rises from -480 to -430 mV, thereby widening the gap between its reduction potential and that of SAM (-1.82 V).7 However, in the presence of the substrate, Lys, the reductive potential drops to -600 mV. The potential of the cluster in the ternary complex is estimated to be in the neighborhood of -990 mV (Figure 1.4). While this is closer to the potential of a generic sulfonium, there remains a substantial mismatch. It is important to note, that in many RS enzymes, additional [4Fe-4S] clusters are necessary to support catalysis. The specific functions that these clusters provide and their reduction potentials (and couples) are an area of active research. 1.10. Models for Homolysis of SAM The mechanism by which SAM is cleaved to generate the central intermediate in all RS enzymes, dAdo•, remains an area of intense interest. There are currently two models for how scission of the S-C5 bond of SAM occurs. 1.10.1. Direct Electron Transfer The simplest mechanism for the activation of SAM is a direct electron transfer event arising from the [4Fe-4S] cluster to the cofactor, which would lead to homolysis of the C-S bond, thereby generating dAdo• and L-Met. A large number of X-ray crystallographic studies show the carboxylate and amino groups of SAM interact directly with the unique Fe of the [4Fe-4S] cluster, and they presented this motif as a defining characteristic across this superfamily.28,34 Moreover, Se-EXAFS using selenium containing Se- adenosyl-L-methionine has shown that the heteroatom is also within a close distance 18 Figure 1.4 – [4Fe-4S] and SAM Redox Potentials Throughout the Catalytic Cycle of LAM. 19 of the cluster.29 Collectively, these suggest all distance requirements necessary to initiate the direct reduction of the SAM by the cluster are met to afford dAdo• production. 1.10.2. Ω Intermediate More recently, Broderick, Hoffman, and coworkers have suggested a different mechanism to generate dAdo• involving the formation of an organometallic intermediate, omega (Ω) (Figure 1.5A).83-85 It has been proposed that SAM is cleaved by covalent attachment of dAdo to the unique iron of the cluster to generate an intermediate that is reminiscent of AdoCbl (Figure 1.5B). The intermediate was first observed in the PFL system. In these experiments, samples containing PFL-AE, PFL, and SAM were frozen, and the cluster was photoreduced. This process leads to a characteristic EPR signal that has been interpreted as representing the organometallic intermediate. Brief heating of the sample to 220 K leads to the disappearance of this signal and the appearance of the glycyl radical on PFL. These results have been interpreted to suggest that Ω is both catalytically and kinetically competent to be an intermediate in the reaction catalyzed by PFL-AE. Similar experiments performed with numerous other radical SAM enzymes including a maturase, an epimerase, and LAM lead to analogous results,84 suggesting that the formation of Ω may be a common feature. 1.11. Scope of Dissertation The focus of this dissertation has been two-fold. In Chapter 2, I will present electrochemical results of SAM cleavage, where for the first time, SAM has been characterized directly in bulk solution. The cathodic peak potential (Epc) of -1.4 V 20 Figure 1.5 – SAM Homolysis Models. A) The Ω intermediate is presumed to form covalent attachment of dAdo to the cluster. B) The structure of AdoCbl. 21 determined in these studies is significantly different from literature estimates, which were all based on reactions carried out with compounds structurally unrelated to SAM and under nonbiological conditions. 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Dong, M.; Horitani, M.; Dzikovski, B.; Pandelia, M. E.; Krebs, C.; Freed, J. H.; Hoffman, B. M.; Lin, H., Organometallic complex formed by an unconventional radical S-adenosylmethionine enzyme. J. Am. Chem. Soc. 2016, 138 (31), 9755-9758. CHAPTER 2 ANALYSIS OF THE ELECTROCHEMICAL PROPERTIES OF S-ADENOSYL-L-METHIONINE AND IMPLICATIONS FOR ITS ROLE IN RADICAL SAM ENZYMES Parts of work have been published previously: Miller, S. A., and Bandarian, V. Analysis of the Electrochemical Properties of S-Adenosyl-L-methionine and Implications for Its Role in Radical SAM Enzymes. J. Am. Chem. Soc., 2019. 114 (28); 11019-11026. 30 2.1 Abstract S-Adenosyl-L-methionine (SAM) is the central cofactor in the radical SAM enzyme superfamily, responsible for a vast number of transformations in primary and secondary metabolism. In nearly all of these reactions, the reductive cleavage of SAM is proposed to produce a reactive species, 5’-deoxyadenosyl radical, which initiates catalysis. While the mechanistic details in many cases are well understood, the reductive cleavage of SAM remains elusive. In this chapter, we have measured the solution peak potential of SAM to be -1.4 V versus the standard hydrogen electrode (SHE) and show that under controlled potential conditions, it undergoes irreversible fragmentation to the 5’-deoxyadenosyl radical (dAdo•). While the radical intermediate is not directly observed, its presence as an initial intermediate is inferred by the formation of 8,5’-cycloadenosine (cyc-dAdo), and by H-atom incorporation into 5’-deoxyadenosine from solvent. Similarly, 2-aminobutyrate is also observed under electrolysis conditions. The implications of these results in the context of the reductive cleavage of SAM by radical SAM enzymes are discussed. 2.2. Introduction Radical SAM enzymes have emerged as a massive superfamily whose functions span primary and secondary metabolism in all domains of life. Recent bioinformatic studies suggest that there may be >100,000 members in the superfamily, many of which are likely to catalyze distinct transformations.1 Despite their ubiquity, the function of only a few handfuls of radical SAM enzymes are known. Significant mechanistic questions still remain, and thus the details surrounding the activation of SAM have remained murky. A comprehensive understanding of the reductive cleavage of SAM by radical SAM 31 enzymes requires that we understand the reduction of SAM as its own entity. It is with this goal in mind that we undertook the voltammetric and amperometric studies of SAM in solution. Frey and colleagues recognized early on with studies on LAM that the energetic requirement to cleave SAM is significant. Model studies available at the time placed the cathodic peak potential (E ) of SAM at ~ -1.6 V (v. SHE), which is significantly more pc reductive than the cluster in its ternary complex poised at -0.99V. If one takes, instead, the experimentally obtained Epc of SAM from this study (~ -1.4 V) as the upper limit for the reduction potential of bound SAM, the difference between the E and the midpoint of the pc potential is still 0.41 V, which still corresponds to a ~40 kJ/mol thermodynamic mismatch. We cannot rule out that binding to the cluster may fine tune the potential of SAM. The measured potential for the reductive cleavage of SAM in solution presented in this chapter provides a starting point for quantitative insights into the mechanism for the activation of SAM by radical SAM enzymes. These data support the notion that, in principle, SAM can undergo reductive cleavage. While the differences between the free energy for cleavage of SAM and the midpoint potential of various iron-sulfur clusters suggest a substantial thermodynamic mismatch, we posit that in fact, nature may have evolved this by design. If the potential of the cluster was such that cleavage of SAM occurred readily, it would potentially lead to formation of significant levels of dAdo•, which in the absence of substrate, could be quenched to form dAdo. Indeed, abortive cleavage of SAM is observed in vitro and hampers radical SAM enzymology. In many of these enzymes, the concentration of their substrate is likely to be significantly less than that of SAM in vivo. By contrast, a mismatch between the midpoint potential of the cluster and Epc of SAM would ensure that cleavage is a rare event that coincides with the presence of 32 bound substrate. 2.3. Results and Discussion The mechanism by which SAM is reductively cleaved in radical SAM enzymes is not well understood. Moreover, to our knowledge, no solution measurements of the redox properties of SAM are available. In this chapter, we carried out voltammetric and amperometric studies to measure the peak potential of SAM under aqueous conditions. We demonstrate that under controlled potential conditions, the cleavage of SAM is a one electron process that leads ultimately to the formation of dAdo and 2-aminobutyrate (2-AB). The implications of these results to understanding the mechanism of cleavage of SAM by radical SAM enzymes in the context of the current mechanistic paradigms are discussed. 2.3.1. Cyclic Voltammetry of Bulk SAM Anaerobic cyclic voltammetry (CV) of SAM was carried out using a three-electrode configuration with a glassy-carbon working electrode in deoxygenated solvents spanning a wide range of dielectric constants (e) to investigate environmental influences on the Epc of SAM. Representative scans are shown in Figure 2.1. In each case, scans in the cathodic direction show an Epc of ~-1.4 to -1.52 V, which, at the lower limit, is over 0.2 V more oxidative than the aqueous reduction of trimethyl sulfonium salts.2 However, regardless of the solvent, all scans in the anodic direction are featureless, indicating that the reduction of SAM is irreversible and correlates with previous observations of sulfonium salt reduction37 and related sulfonamides.8 In control experiments we find a linear relationship between 33 Figure 2.1 – Cyclic Voltammograms of SAM in (a) water at 120 mV/sec, (b) acetonitrile at 110 mV/sec, (c) ethanol; at 85 mV/sec, and (d) tetrahydrofuran at 100 mV/sec. Final concentrations of SAM were 2mM and 100mM KI as the supporting electrolyte. 34 the height of the peak current (ip) and the square root of scan rate (see Figure 2.2), indicating that the reduction of SAM is limited by diffusion. To ensure the small solventdependent differences in the peak potentials are significant, after each set of scans, a CV of methyl viologen (MV) (Figure 2.3) was obtained following numerous scans at various scan rates (such as those shown in Figure 2.4). These controls show that the midpoint potentials and peak separation values of the methyl viologen controls remain internally consistent. Finally, the reductive peak potential of SAM is not sensitive to the identity of the counter ion (potassium bromide (KBr), potassium chloride (KCl), or potassium iodide (KI)), or to the source of SAM (synthetic or commercial (Figure 2.5). The CV profiles and peak potentials are independent of the supporting counter ion or the stock of SAM. Therefore, in all subsequent experiments, we employ KI as the supporting counter ion and commercially obtained SAM. Previous electrochemical studies of sulfonium and iodinium salts demonstrated 7 9 that covalent functionalization of the glassy carbon electrode can occur upon reductive cleavage. To determine if covalent attachment to the surface was occurring, a solution of SAM in acetonitrile was subjected to iterative CV scans. The measured Epc remains at 1.4 V (Figure 2.6 i,ii). Upon applying a potential of -1.6 V for 300 s, the Epc moves towards a more oxidative position of -1.2 V (Figure 2.6 iii), which in subsequent scans, slowly trends towards the initial value of -1.4 V (Figure 2.6 iv,v). These results support the transient, reversible association of SAM to the glassy carbon working electrode surface during electrolysis. Therefore, the measured peak potentials are not significantly affected by surface passivation. 35 Figure 2.2 – Dependence of SAM Reductive Peak Current on Scan Rate. Plots of peak potentials from CV of SAM in various solvents from Figure 2.4. The reactions were carried out in (circle) water, (square) ethanol, (triangle) acetonitrile, and (diamond) tetrahydrofuran. 36 Figure 2.3 – Cyclic Voltammograms of Methyl Viologen (MV). All data were collected using a 0.1 mM MV, 0.2 M KCl solution with a scan rate of 100 mV/sec using a glassy carbon working electrode that had previously been utilized to determine scan rate dependence measurements with SAM (see Figure 2.4) in (A) water, (B) ethanol, (C) acetonitrile, and (D) tetrahydrofuran. Peak separation (DEp) and midpoint potential (E ’) versus S.H.E. for MV are as follows: (A) DEp= 66 mV, E ’= -438 mV (B) DEp= 66 mV, E ’= -438 mV; (C) DEp= 64 mV, E ’= -444 mV; and (D) DEp= 64 mV, E ’= -438 mV. o o o o o 37 Figure 2.4 – Solvent and Scan Rate Dependence of Reductive Wave and Currents in CV Experiments with SAM. The reactions were carried out as described in the Experimental Procedures in (A) acetonitrile, (B) ethanol, (C) water, and (D) tetrahydrofuran. The peak currents are plotted in Figure 2.2. 38 Figure 2.5 – Cyclic Voltammetry of Commercial (gray) and Enzymatically Prepared SAM (black) in (i) 0.1 M KBr, (ii) 0.1 M KCl, and (iii) 0.1 M KI. All scans were collected at a scan rate of 140 mV/sec using a glassy carbon working electrode. 39 Figure 2.6 – Glassy-Carbon Working Electrode Functionalization. Electrode functionalization was tested by obtaining CV data for 2 mM SAM in 95% acetonitrile using iterative scans; i) is the first sweep after mixing SAM. At this point, the sample was swept twice, and ii) represents the third sweep. After these initial sweeps, a potential of -1.6 V was applied for 300 s and followed by a fourth sweep (iii). Following two more sweeps of the sample, trace iv was obtained. Trace v) was obtained after two additional sweeps. 40 2.3.2. SAM Is Cleaved Under Reducing Conditions to Produce dAdo We next examined the products of reductive cleavage of SAM. In these experiments, SAM and the supporting electrolyte KI were mixed under anaerobic conditions. The reactions were poised at potentials ranging from -0.8 to -2.8 V, and aliquots were withdrawn at various times after initiating the reduction. dAdo is readily detected by liquid chromatography-mass spectrometry (LC-MS) analysis of the samples (Figure 2.7A). The rate of formation of dAdo at each potential was determined by quantifying the peak area of dAdo formed during electrolysis by comparing it to a known dAdo standard curve. Figure 2.7B shows a representative example of the rate data at -1.5 V. Plots of the rate of formation of 5’-dAdo versus cell potential (Figure 2.7C) reveal a clear midpoint potential for the cleavage of SAM, which is centered at -1.4 V. This value agrees remarkably well with data obtained from the CV experiments. We note that the 5 mM SAM used in all these measurements was sufficient to achieve the maximal cleavage rate under the experimental conditions. The reductive cleavage of SAM generates Met and dAdo•, which is presumably quenched in solution by a hydrogen atom derived from a solvent exchangeable site. We note that in enzyme catalyzed reactions, dAdo is often formed even in the absence of substrate due to abortive cleavage cycles, which experiments in deuterium oxide (D2O) have shown can be quenched by solvent exchangeable sites. The MS of dAdo exhibits a major base peak at a mass to charge ratio (m/z) of 252, which is shown in Figure 2.8A. The relative abundances of the base peak and the corresponding +1 and +2 natural abundance isotope peaks are 100% to 10.8% and 0.6%, respectively. These are consistent with the theoretical values. By contrast, when the same experiment is carried out in 95% 41 Figure 2.7 – SAM Is Cleaved Under Reducing Conditions to Produce dAdo. (A) UHPLC trace of authentic dAdo standard (upper), extract ion chromatogram (EIC) of m/z 252.11 corresponding to dAdo from authentic standard (middle), and dAdo from electrolysis of SAM (lower). The small difference in retention times observed between UV and MS is the result of the distance between the UV-visible detector and the inline MS analyzer. (B) Representative dAdo versus time plot at – 1.5 V. dAdo standards were used to quantify dAdo. (C) Rate of dAdo as a function of cell potential reveals a midpoint of ~ -1.4 V versus SHE. 42 Figure 2.8 – Isotope Incorporation from Bulk Solvent into dAdo. Isotopic peak distribution of dAdo when electrochemically reduced in (A) H2O or (B) D2O. 43 D2O, in addition to the major base peak, a substantial peak at m/z of 253.1145 (Figure 2.8B), which is within 5 parts per million (ppm) of that expected for dAdo containing a single deuterium atom. The source of this deuterium is not known, but we presume it is derived from a solvent exchangeable site. We also see unlabeled dAdo, albeit at much smaller amounts, which could arise from non-exchangeable sites, such as the buffer, or exchange with a small amount of H2O. These data unambiguously establish dAdo is a product of the controlled potential reduction of SAM. 2.3.3. dAdo Forms in a One Electron Process While deuterium incorporation into dAdo suggests that dAdo• forms in the reductive cleavage of SAM, it can also undergo a secondary reduction, followed by protonation to produce dAdo. Frey and Abeles demonstrated that when adenosylcobalamin (AdoCbl) is photolyzed under anaerobic conditions, 8,5’-cycloadenosine (cyc-dAdo) is one of the observed products.10-11 The formation of cyc-dAdo presumably occurs through a radical addition between the unpaired electron at the 5’-position and the C-8 of the base. We reasoned that observation of cyc-dAdo would implicate the formation of dAdo• during controlled potential electrolysis of SAM. As an initial control, we carried out anaerobic photolysis of AdoCbl and analyzed samples before and after complete cleavage of the molecule. In our experimental setup, exposure of a 0.1 mM solution of AdoCbl in water to light for 30 min leads to the disappearance of the AdoCbl features at 520 nm and the appearance of cob(II)alamin at 475 nm (Figure 2.9A). We observe no oxidation of the cob(II)alamin, which is reasonable considering that oxygen is excluded in these experiments. As expected, LC-MS analysis of the reaction mixture before and after 44 Figure 2.9 – Analysis of Photolysis Products of AdoCbl. (A) UV-visible spectra of 0.1 mM AdoCbl solution during photolysis. (B) EIC traces at m/z 252.11 AdoCbl prior to (gray) and resulting from photolysis (black) (upper), and EIC trace at m/z 250.09 prior to (gray) and resulting from photolysis (black) (lower). (C) HCD fragmentation of authentic dAdo standard (upper; RT 12.2 min, m/z 252.11) and authentic cyc-dAdo (lower; RT 10.6 min, m/z 250.09). (D) HCD fragmentation of dAdo (upper; RT 12.2 min, m/z 252.11) and cyc-dAdo (lower; RT 10.6 min, m/z 250.09) from photolysis of AdoCbl. 45 exposure to light shows that the homolysis leads to the formation of dAdo, which has the same retention time and mass (m/z=252) of that formed in the controlled potential experiments with SAM (compare Figure 2.9B upper trace and Figure 2.7A). Formation of dAdo requires light and did not form if the sample was not photolyzed. To identify the presence of cyc-dAdo, the extracted ion chromatograms from samples that were removed before and after photolysis of AdoCbl were examined for species with m/z of 250. The extracted ion chromatogram (EIC) reveals two peaks (at 3.8 and 10.6 min) (Figure 2.9B), which are only observed under illumination. As discussed below, the 10.6 min peak corresponds to cyc-dAdo. To definitively identify the10.6 min peak as cyc-dAdo comparative MS/MS analysis of dAdo and an authentic cyc-dAdo standard were carried out. High-energy collisional dissociation (HCD) of dAdo releases adenine (Ade), which has a m/z of 136.0614 (Figure 2.9C upper trace). By contrast, fragmentation of cyc-dAdo only leads to loss of the water resulting in a m/z of 232.08. We do not observe release of the base at any HCD power setting examined (Figure 2.10) with the cyc-dAdo standard; this is presumably because fragmentation at both the N-glycosidic bond and the C5’-C8 crosslink are unlikely (Figure 2.9C lower trace and Figure 2.10). Analysis of the species eluting at 10.6 min with m/z of 250 formed during photolysis of AdoCbl reveals identical fragmentation properties (Figure 2.9D lower trace). The HCD fragmentation of the peaks corresponding to dAdo and cyc-dAdo observed during the photolysis of adenosylcobalamin are identical to the authentic standards (compare Figures 2.9C and 2.9D). Since the peak at 3.8 min did not have retention time or fragmentation pattern of cyc-dAdo, we did not probe the identity of this species further. 46 Figure 2.10 – Fragmentation of Authentic cyc-dAdo Standard in the HCD Cell at Various Power Settings. The peak at m/z of 250.09 peak fragments to a species with m/z of 232.08 at increasing power. 47 The extracted ion chromatograms from controlled potential electrolysis of SAM also reveal formation of cyc-dAdo (Figure 2.11), which elutes with exactly the same retention time as samples from photolysis of AdoCbl and the authentic standards (compare Figure 2.9B and Figure 2.12). Moreover, when subjected to HCD fragmentation only loss of water, rather than the release of the adenine base (Figure 2.11B), is observed. This is consistent with its assignment as cyc-dAdo (Figure 2.9C and Figure 2.9D). 2.3.4. Reductive Cleavage of SAM also Produces 2-aminobutyrate The results described above clearly demonstrate that electrolysis of SAM produces 5’-dAdo•. However, in a small subset of radical SAM enzymes, homolysis of SAM is also able to produce a 2-AB radical. For example, 2-AB, which is presumably formed by the reductive cleavage of SAM, is used to posttranslationally modify a histidine residue in elongation factor-2 (EF-2) forming “diphthamide.”12,13 Therefore, we investigated the production of 2-AB in controlled potential electrolysis of SAM. 2-AB is readily detected by LC-MS analysis using a hydrophilic high-pressure liquid chromatography (HPLC) column (Figure 2.13A, upper trace). The species eluting at 5.7 min exhibits a m/z of 104.0707), which is within 3.8 ppm of the theoretical mass of 2-AB. Electrolysis of SAM under the conditions needed to produce dAdo also form a species with identical retention time (5.7 min) and m/z (104.0707) as the standard (see Figure 2.13A and 2.13B, middle trace). The m/z of the species observed in the electrolysis experiments is also within 3.8 ppm of the theoretical mass. As with dAdo, when this experiment is carried out in D2O an additional peak at m/z 105.0769 (Figure 2.13B, lower trace) is observed. The observed m/z is within 3.8 ppm of the expected value for incorporation of a single deuterium (D) into 2- 48 Figure 2.11 – Characterization of m/z 250 Species from Controlled Potential Electrolysis of SAM. (A) EIC at m/z 250.09 of electrolysis of SAM at -1.8 V. (B) Fragmentation of the species at m/z of 250.09 produces fragments that are identical to those observed with cyc-dAdo standards (Figure 2.9B). 49 Figure 2.12 – EIC Trace at m/z of 250 for a cyc-dAdo Standard. The separation was carried out as described in the Experimental Procedures. 50 Figure 2.13 – Controlled Potential Electrolysis of SAM Produces 2-AB. Controlled potential electrolysis experiments were carried out at -1.8 V. (A) The EIC traces correspond to 2-AB standard (upper trace, m/z=104.07), electrolysis in H O (middle trace, m/z=104.07), and electrolysis in D O (lower trace, m/z=105.07). (B) Mass spectra corresponding to the peak at 5.7 min in EIC traces shown in panel A. (C) MS/MS analysis of the base peaks in spectra shown in (B). 2 2 51 AB. As with dAdo, 2-AB appears to quench by incorporating hydrogen (H) from both solvent exchangeable and non-exchangeable sites. To unambiguously establish that the 2-AB observed in these experiments is identical to the standard, the base peaks were subjected to MS/MS analysis (Figure 2.13C). The major fragmentation product of the 2-AB standard and 2-AB produced in H2O is at m/z of 58.0659, which is consistent with the loss of the carboxylate. In D2O, peaks at 105.07 and 59.07 are observed, consistent with incorporation of deuterium into 2-AB. To establish if the rate of cleavage of SAM to produce 2-AB correlates with the peak potential of SAM, controlled potential experiments were conducted where the cell was poised at -1.25, -1.5, or -2.25 V (Figure 2.14A), followed by simultaneous quantification of dAdo and 2-AB. Because 2-AB does not have a UV-visible feature that can be used for quantitation, we integrated the peak area for 2-AB and dAdo in the EIC traces of these species at various times and quantified by comparison to known standards. As shown in Figure 2.14B, both species are formed at similar rates over the range of potentials examined. Indeed, the dependence of cleavage rate on the applied potential is very similar to that shown for dAdo alone (compare Figure 2.7C and Figure 2.14B), with a midpoint for rate occurring near the peak potential of SAM. Therefore, at least to a first approximation, there is no energetic preference for which C-S bond is cleaved, and the choice of cleavage in enzyme-catalyzed reactions is dictated by the local environment of the active site. 52 Figure 2.14 – dAdo and 2-AB Are Produced with Similar Rates During Controlled Potential Electrolysis of SAM. In these experiments solutions were poised at -1.25V (circle), -1.5V (triangle), or -2.25V (square). Samples were withdrawn at various times and (A) dAdo (black) and 2-AB (red) were quantified on the basis of the area of the EIC peak. (B) The rate of formation of dAdo (black) and 2-AB (red) versus the cell potential. 53 2.4. Conclusions In summary, the results presented here demonstrate that the controlled potential electrolysis of SAM produces both dAdo and 2-AB with an Epc of less than -1.4 V. In the enzyme, the choice of dAdo• or 2-AB• is presumably dictated by active site constraints. We note in passing that while the difference between the Epc of SAM (-1.4 V) and those of alkyl and aryl sulfoniums may seem small, the value reported here is essential to ground any future discussion of the mechanisms by which radical SAM enzymes overcome substantial thermodynamic mismatches to allow the radical cluster to catalyze the cleavage of a C-S bond of SAM. To our knowledge, these data are the first measurement of the reductive potential of SAM and evidence for its ability to produce dAdo• or 2-AB• under enzyme-free conditions. 2.5. Materials and Methods 2.5.1. Enzymatic Preparation of SAM Synthesis and purification of SAM was performed as previously described.14 2.5.2. Cyclic Voltammetry of SAM All solvents were cycled into a Coy anaerobic chamber (maintained at 95%N /5%H ) and deoxygenated overnight. The reaction mixtures were prepared by 2 2 combining an aliquot (50 µL) from an aqueous stock solution of SAM (Sigma) with an equal volume of a 2 M solution of KI. The solution was vigorously stirred before adding 0.45 mL of solvent. For the reactions performed in acetonitrile or ethanol, it was necessary to include 50 µL of water to solubilize SAM and KI. Prior to each scan, a 3 mm glassy- 54 carbon working electrode (CH Instruments) was polished with 0.05 µm alumina. Next, the working, calomel reference (CH Instruments), and platinum counter electrodes (Pine Research) were submerged into the solution. Voltammetry was performed using a Model 1200C hand-held potentiostat (CH Instruments), with a scan window of -0.05 to -1.6 or -1.85 V. Following each set of CV scans, a 0.2 mM aqueous solution of methyl viologen (MV) containing 0.1 M KCl was scanned at a rate of 100 mV/sec as a control. All voltammetric measurements were corrected to the standard hydrogen electrode potential (S.H.E). 2.5.3. Controlled Potential Electrolysis of SAM The reaction mixtures contained 5 mM SAM, 50 mM PIPES•NAOH (pH 7.4), and 0.2 M KI in a total volume of 1 mL. The solution was stirred to mix the components. Prior to initiating the reaction, mixing was stopped and prepolished 3 mm glassy carbon working, calomel reference, and platinum counter electrodes were submerged in the solution. An aliquot of the solution (70 µL) was withdrawn as a pre-electrolysis standard, and the reaction was initiated by poising the cell at the desired potential. An aliquot (70 µL) of the mixture was withdrawn at various times after initiating the reaction. The solution was stirred after each withdrawal, but the stirring was off during the electrolysis. All aliquots from the reaction were frozen at the end of the experiment. For the reactions performed in D O all components were dissolved in D2O except the 1 M PIPES•NaOH (pH 7.4) stock 2 solution. The final D2O content of the reaction mixture was ~95%. 55 2.5.4. Analysis of Products of Controlled Potential Electrolysis of SAM An aliquot (30 µL) of each of the time points was analyzed with a Vanquish UHPLC (Thermo Fisher) with a diode array detector, which had been interfaced to a QExactive mass spectrometer to obtain in-line mass spectrometric data of all species. The separation of hydrophobic products was performed on a Hypersil Gold C-18 column (2.1×150 mm, 1.9 μm particle size) column with a 11.5 min gradient of 0-30% acetonitrile in 0.1% aqueous TFA. The dAdo was quantified by comparing the peak area to dAdo standards. Detection and quantification of the 2-aminobutyrate product was achieved by mixing an aliquot (20 µL) with 80 µL of acetonitrile and analyzed on a SeQuant ZiccHILIC column (2.1x100mm, 3 μm particle size) over a 30 min gradient from 20% Buffer B to 67% Buffer B in Buffer A (Buffer A: 90% ACN, 10% 25 mM ammonium acetate; Buffer B: 10% ACN, 90% 25mM ammonium acetate). 2-AB was quantified by comparing the EIC peak area to those obtained from a standard curve. All MS measurements were performed in the positive ion mode with a resolution setting of 100,000 and m/z range of 50 to 650. MS/MS was performed in by fragmentation in the HCD cell of the instrument at various power settings, as noted in the results. 2.5.5. Analysis of Homolysis of 5’-deoxyadenosylcobalamin (AdoCbl) An aqueous 0.1 mM solution of 5’-deoxyadenosylcobalamin was prepared in the anoxic chamber. The solution was transferred to an anaerobic cuvette and removed from the chamber. A UV-visible spectrum was recorded using an Agilent 8453 diode array spectrometer and homolysis was initiated using a table top lamp. Spectra of the sample 56 were recorded at several points during photolysis. A sample was withdrawn at the end of 30 min, at which point homolysis was complete. The LC-MS/MS analysis of the preand posthomolysis samples was carried out as described above for the controlled potential experiments. 2.6 References 1. Holliday, G. L.; Akiva, E.; Meng, E. C.; Brown, S. D.; Calhoun, S.; Pieper, U.; Sali, A.; Booker, S. J.; Babbitt, P. C., Atlas of the radical SAM superfamily: divergent evolution of function using a "plug and play" domain. Methods Enzymol. 2018, 606, 1-71. 2. Colichman, E. L.; Love, D. L., Polarography of sulfonium salts. J. Org. Chem. 1953, 18 (1), 40-46. 3. Saeva, F. D.; Morgan, B. P., Mechanism of one-electron electrochemical reductive cleavage reactions of sulfonium salts. J. Am. Chem. Soc. 1984, 106 (15), 4121-4125. 4. Kampmeier, J. A.; Hoque, A. K.; Saeva, F. D.; Wedegaertner, D. K.; Thomsen, P.; Ullah, S.; Krake, J.; Lund, T., Regioselectivity in the reductive bond cleavage of diarylalkylsulfonium salts: variation with driving force and structure of sulfuranyl radical intermediates. J. Am. Chem. Soc. 2009, 131 (29), 10015-10022. 5. McKinney, P. S.; Rosenthal, S., The electrochemical reduction of the triphenylsulfonium ion. J. Electroanal. Chem. Interfacial Electrochem. 1968, 16 (2), 261-270. 6. Finkelstein, M.; C. Petersen, R.; D. Ross, S., Electrochemical degradation of aryl sulfonium salts. J. Electrochem. Soc. 1963, 110 (5), 422-425. 7. Vase, K. H.; Holm, A. H.; Norrman, K.; Pedersen, S. U.; Daasbjerg, K., Electrochemical surface derivatization of glassy carbon by the reduction of triaryl- and alkyldiphenylsulfonium salts. Langmuir 2008, 24 (1), 182-188. 8. Viaud, P.; Coeffard, V.; Thobie-Gautier, C.; Beaudet, I.; Galland, N.; Quintard, J. P.; Le Grognec, E., Electrochemical cleavage of sulfonamides: an efficient and tunable strategy to prevent beta-fragmentation and epimerization. Org. Lett. 2012, 14 (3), 942-945. 9. Vase, K. H.; Holm, A. H.; Norrman, K.; Pedersen, S. U.; Daasbjerg, K., Covalent grafting of glassy carbon electrodes with diaryliodonium salts: new aspects. Langmuir 2007, 23 (7), 3786-3793. 57 10. Frey, P. A.; Essenberg, M. K.; Abeles, R. H., Studies on the mechanism of hydrogen transfer in the cobamide coenzyme-dependent dioldehydrase reaction. J. Biol. Chem. 1967, 242 (22), 5369-5377. 11. Hogenkamp, H. P. C., A cyclic nucleoside derived from coenzyme B12. J. Biol. Chem. 1963, 238 (1), 477-480. 12. Zhang, Y.; Zhu, X.; Torelli, A. T.; Lee, M.; Dzikovski, B.; Koralewski, R. M.; Wang, E.; Freed, J.; Krebs, C.; Ealick, S. E.; Lin, H., Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme. Nature 2010, 465 (7300), 891-896. 13. Zhu, X.; Dzikovski, B.; Su, X.; Torelli, A. T.; Zhang, Y.; Ealick, S. E.; Freed, J. H.; Lin, H., Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis. Mol. Biosyst. 2011, 7 (1), 74-81. 14. McCarty, R. M.; Krebs, C.; Bandarian, V., Spectroscopic, steady-state kinetic, and mechanistic characterization of the radical SAM enzyme QueE, which catalyzes a complex cyclization reaction in the biosynthesis of 7-deazapurines. Biochemistry 2013, 52 (1), 188-198. CHAPTER 3 MECHANISM OF ACTIVATION FOR A CHIMERIC RADICAL SAM ENZYME-FLAVODOXIN FUSION PROTEIN Respective contributions: The wild type fusion construct plasmid used in the experiments presented in this chapter was prepared by Dan Dowling and Ben Bell in our collaborator Cathy Drennan’s lab at MIT. All other experiments were carried out at the University of Utah. 59 3.1 Abstract Members of the expansive radical SAM (RS) superfamily leverage the reductive cleavage of S-adenosyl-L-methionine to perform complex radical-mediated transformations. 7-Carboxy-7-deazaguanine (CDG) synthase (QueE), performs a challenging radical mediated ring contraction step on its substrate 6-carboxy-5,6,7,8,tetrahydropterin (CPH4) to generate CDG, which is a precursor for all deazapurine containing natural products. QueE reductively cleaves SAM using a [4Fe-4S] cluster, which in turn must be reduced from the resting +2 oxidation state to the +1 state to support cleavage. While the biological reductant is not known, it is thought that flavodoxins are potential reductants, though small molecule reductants, such as dithionite, can also function in reducing the cluster. In this chapter, we examine the properties of a chimeric enzyme, where the N-terminus of the Escherichia coli (E. coli) QueE has been fused to the “generic” flavodoxin A (FldA) protein from the same organism. While the chimeric protein is catalytically active, the activity that is observed is lower than that observed with equivalent concentrations of QueE and FldA monomers. The data are consistent with activity arising from intermolecular activation between chimeric monomers. 3.2 Introduction Pyrrolopyrimidine nucleosides are widely distributed in nature. The 7-deazapurine core of these compounds is present in such disparate molecules as tRNA, DNA, and as secreted secondary metabolites produced by various strains of Streptomyces.1-2 While the first deazapurine, toyocamycin,3 was discovered over 40 years ago, the biosynthetic pathway that produces these compounds had remained elusive until the 2000s. 60 Early radiolabeling studies carried out by Nishimura, Suhadolnik, and colleagues 47 had pointed to a purine as the precursor to the deazapurine. The data suggested that substantial rearrangements of the purine core must occur, which retain C-2 but eliminate C-8. Moreover, carbon atoms in the ribose moiety were shown to be incorporated into the natural product. The biosynthetic pathway to the deazapurines was reconstituted by the Bandarian lab with the discovery of the toyocamycin biosynthetic cluster.8-9 The current paradigm for the biosynthesis of toyocamycin, sangivamycin, and the related modified tRNA base queuosine is shown in Figure 3.1. The key step in the pathway, conversion of the purine to a deazapurine, is catalyzed by7-Carboxy-7-deazaguanine (CDG) synthase (QueE), which converts 6-carboxy-5,6,7,8,-tetrahydropterin (CPH4) to the CDG product. QueE is a member of the radical SAM (RS) superfamily. Biochemical and structural studies have shown that QueE houses a [4Fe-4S] cluster, which binds and activates S-adenosyl-Lmethionine (SAM).8, 10-12 The reductive cleavage of SAM generates 5’-deoxyadenosyl radical (dAdo•), which initiates the catalytic cycle. The reaction catalyzed by QueE is formally a radical-mediated ring contraction. Biochemical studies with deuterated isotopologues of the substrate have shown that dAdo• abstracts a H-atom from C-6 to generate a substrate radical (see Figure 3.2).8 The initially formed radical undergoes rearrangement and quenching to generate a gem-amino carboxylate, which subsequently undergoes stereoselective deprotonation to eliminate ammonia and form CDG. A large number of X-ray crystal structures of the protein solved in collaboration between the Bandarian and Drennan labs 10-12 support many aspects of this mechanism. 61 Figure 3.1 – Biosynthetic Pathway to 7-deazapurine tRNA Base Queuosine. QueE catalyzes a key step during the conversion of purine to 7-deazapurine. Dashed lines indicate multiple steps. 62 Figure 3.2 – Radical Mediated Ring Contraction Mechanism of QueE. C-6 position (cyan), C-7 (magenta). Dashed lines indicate multiple steps. 63 Previous investigations into the biological and chemical activation of radical SAM proteins has revealed reductive preferences as diverse as their functions. In the earliest example of this, the pyruvate formate lyase activating enzyme (PFL-AE) was shown by Knappe et al.13 to prefer a biological reduction system. Despite the utilization of a noncognate E. coli flavodoxin, FldA, this work set precedent for the utility of a biological reduction system in vitro. Conversely, work performed by Grove et al. on two anaerobic sulfatase enzymes, AtsB and anSME, demonstrated chemical reduction by dithionite to be a more robust reductant, indicated by a 10-100 fold increase in activity when compared to the E. coli reduction system.14-15 Similar results were obtained for the Burkholdeira multivorans (BmQueE) homolog 11 when dithionite instead of EcFldA was used. However, these insights cannot be generalized to predict when DT or a Fld may be preferred, and whether the species from which the Fld is derived makes a difference. The key to the ring contraction catalyzed by QueE is a reductive cleavage of SAM to generate the dAdo•. In vitro turnover by QueE is supported by dithionite.11, 16 While the reducing system is not known in vivo, it is thought that flavodoxin (Fld) and flavodoxin reductase (Fpr) deliver reducing equivalents from NADPH to the protein in one-electron processes. Indeed, it has been shown that at least with the Bacillus subtilis protein, both Fld homologs of the organism can support turnover by the B. subtilis QueE (BsQueE) at significant levels. Interestingly, the Fpr/NADPH can be replaced by dithioninte, which is capable of reducing Fld, which in turn reduces the BsQueE.16 Many organisms contain multiple copies of Fld suggesting these activating proteins perform nonredundant roles in initiating redox chemistry. Flavodoxins are small (~20 kDa) FMN-binding proteins with a common Rossman-like fold despite sharing low sequence 64 homology.17 While numerous structures of flavodoxins alone have been determined,18-27 no structure has been solved bound to a known target RS protein. Therefore, the orientation, induced binding movements, and distances involved in facilitating electron transfer to the [4Fe-4S] cluster are all outstanding questions in the field. Indeed, in the experiments where it was shown that the BsQueE could be reduced by Fld from the same organism, one of the two Fld homologs was more effective.16 The details of the reductive mechanism of RS enzymes remains an area of intense contemporary research. In all RS enzymes, the [4Fe-4S] cluster is positioned towards the lateral opening of the partial TIM barrel with differential electrostatic landscapes surrounding each active site 10-12 (reviewed in Broderick et al. 2014).28When compared to the structures of known flavodoxins from the corresponding organisms, electrostatic complementarity is sometimes apparent.12 The current working model is that these structural differences are vital in determining the biological reduction preferences. However, there is very little information on the interaction. In another complication, some organisms harbor RS proteins but not Fld.29 In at least some cases, ferredoxins can perform reduction of the [4Fe-4S] cluster in vitro. Notably, in a screening of five ferredoxin homologs, those with a single [4Fe-4S] cluster that was readily reduced by either Fpr/NADPH or dithionite were consistently more successful in activating the RS methylthiotransferase, MiaB, from the same organism.30 While the necessity for reductive activation would suggest that all RS enzymes must be intimately coupled to some form of a reducing system, there are very few examples of RS enzymes that carry their own activation “toolkit.” Perhaps the best-known example is the TYW1 protein from mammalian sources, which at least based on sequence similarity 65 appears to be composed of two domains – a RS domain coupled to a Fld-like one.31 However, this protein has not been characterized to date. We sought a model system to use in studying the reductive activation of QueE. To this end, we have generated a chimeric QueE where the Escherichia coli QueE homolog, EcQueE, is fused to the EcFldA. This chapter reports biochemical and kinetic studies of this chimeric protein. 3.3 Results and Discussion As purified members of the RS superfamily are catalytically inactive in the absence of a reductant to reduce the bound [4Fe-4S] cluster that activates SAM from its +2 to active +1 state. In vitro reductants include small molecules such as dithionite or titanium citrate. However, in the cell, this task is likely accomplished by flavodoxin or ferredoxin proteins. To gain further insights into the reductive activation process, we have examined a chimeric protein, where the EcQueE is fused to the FldA protein from the same organism. 3.3.1. Purification and Cofactor Analysis The construct containing N-terminal hexahistidine tagged FldA and EcQueE fusion (ECF) was obtained by placing the fldA gene of E. coli upstream of the queE from the same organism. The construct encoded a (GGGGS)5 linker between the two. The fusion construct was transformed into BL21(DE3) cells along with the pBD1282 plasmid, which encodes proteins that are involved in the assembly and insertion of the [4Fe-4S] clusters in nitrogenase.32 All purification steps were carried out under anoxic conditions, as described in Materials and Methods. 66 The purified proteins were reconstituted anaerobically with Fe, S, and FMN. The purity of the resulting ECF was estimated to be at least 95% via SDS-PAGE (Figure 3.3A). EcQueE was expressed, purified, and reconstituted in a similar manner (Figure 3.3B). An additional construct, ECF∆RS where point mutations (C31A, C35A, and C38A) of the [4Fe4S] cluster binding residues caused loss of RS cluster binding. The ECF∆RS variant appears to be partially degraded, presumably because it is destabilized (Figure 3.3C) Purification of FldA was carried out in a single step using a DEAE-Sepharose column aerobically at 4°C as previously described then anaerobically desalted (Figure 3.3D). The UV-visible spectra of various proteins are shown in Figure 3.4. The spectra of FldA (ii), and ECF∆RS (iii), and ECF (iv) are nearly identical and are dominated by the flavin features (i) at 467nm, masking the broad 420-550nm region that is characteristic of EcQueE (v). The FMN content of ECF and ECF∆RS was determined by acid precipitation followed by LC-MS analysis. The data show that ECF and ECF∆RS harbor equimolar quantities of FMN per monomer (Table 3.1). Iron and sulfide analysis carried out with ECF shows near full reconstitution of the [4Fe-4S] cluster and complete loss of both components in the ECF∆RS mutant. We were unable to achieve full reconstitution of the EcQueE. Therefore, in the foregoing discussion, all experiments are carried out using the known iron content and not the amino acid concentration. 3.3.2. ECF Is Catalytically Active To determine whether the fusion protein is catalytically active, anaerobic steadystate kinetic measurements were carried in the presence of varying concentrations of dithionite (DT) or flavodoxin reductase (Fpr) to compare the efficiency of chemical 67 Figure 3.3 – Characterization of Protein Purity. SDS-PAGE determination of protein purity A) E. coli FldA-QueE Fusion, B) E. coli QueE, C) ECF∆RS, and D) FldA. 68 Figure 3.4 – UV-Visible Overlay of Purified Proteins. i) FMN (yellow), ii) FldA (red) , iii) ECF∆RS (gray), iv) ECF (black, solid), and v) QueE (black, dashed). 69 Table 3.1 – Cofactor Quantification of Purified Proteins. N.D.= not determined 70 versus biological reductants, respectively. To ensure activation of the protein, all components of the assay were incubated for 10 min prior to the addition of CPH4 to initiate the turnover. Aliquots (50 µL) were withdrawn at various times after mixing, quenched by mixing with TCA, and CDG was quantified by measuring the area under the peak in extracted ion chromatograms at m/z 195 corresponding to CDG. Peak areas measured with CDG control samples served as standards. Control experiments show that the rate of formation of CDG is linear up to 4 min; therefore, rates were measured based on the 4 min endpoint and plotted against the concentration of DT or FPR (see Figure 3.5). The data were fit to the Michaelis-Menten equation to extract maximal rate and half-maximal concentrations. With DT as the reductant, the maximal rate that is achieved is ~ 1.0 ± 0.1 min-1. By contrast, with FPR, the activity is more modest at 0.19 ± 0.02 min1 . We were surprised by the differential level of overall turnover with the FPR/NADPH versus DT, and reasoned that perhaps with FPR/NADPH, the presence of the tethered QueE may limit binding of FPR to the enzyme. Therefore, the experiments with ECF were repeated in the presence of 0.5 µM methyl viologen (MV). We reasoned that MV would serve to relay reducing equivalents between FPR/NADPH and Fld and/or QueE. Indeed, methyl viologen (MV) was able to elevate CDG production to a similar extent as chemical reduction (compare Figure 3.5A and Figure 3.5B gray points). These results suggest the FPR to FldA electron transfer event may be a rate limiting step in the activation of ECF. The results show that a modest (3-fold) increase is observed. More significantly, in the presence of MV the maximal activity is achieved at the lowest concentration of FPR tested (0.1 µM), supporting the notion that in the absence of the mediator, the rate limiting step is likely the binding of FPR to Fld, which has been reported previously.33 71 Figure 3.5 – Chemical and Biological Reduction of ECF. Initial rates (v0 ) of CDG production as a function of A) DT and B) FPR (black) or FPR + MV (gray). Plots are an average of three experiments. Note the break in axis in panel B. 72 3.3.3. Is Activation of ECF Facilitated by Intermolecular or Intramolecular Reduction? The data presented above show that ECF is catalytically active in the presence of DT. However, the data do not show whether the activation is due to the tethered Fld reducing the QueE in the chimera, or one that is on a different polypeptide, or both. To begin to distinguish between these we reasoned that it may be possible to disentangle these possibilities by examining the extent to which MV can enhance the activity beyond what is obtained with DT alone. The concentration of DT was kept at 25 mM, which studies shown in Figure 3.4A show is sufficient to achieve maximal activation of ECF. Rates were measured by quantifying the [CDG] as described above over a 4 min incubation, during which formation of the product is linear with time (Figure 3.6). Identical reactions were also carried out with EcQueE. In the absence of MV, both EcQueE and ECF exhibit similar activities (0.6 ± 0.05 v 0.8 ± 0.07 min-1). Interestingly, when 0.5 µM MV is added, the rate of production of CDG increases to 3.6 ± 0.4 min-1 with EcQueE, but 1.8 ± 0.1 min1 with ECF. While these are comparable, there is reproducibly more activity with the EcQueE. Nevertheless, the fact that both enzymes have essentially identical rates in the absence of MV suggests that much of the activation that is observed in the presence of reductant is likely from trans rather than cis interactions. One would have expected a substantial difference between the activities of QueE and ECF in the absence of any mediators, and no effect of the mediator on the ECF if the activation resulted from direct intramolecular interaction between the two domains of a single polypeptide. To gauge the extent to which activity of the EcQueE is enhanced with a biological reducing system, the rates above were compared to ones when FldA is added to the reaction 73 Figure 3.6 – Progress Curves for Formation of CDG. CDG concentrations detected in reaction mixtures containing ECF (cyan/blue), QueE alone (gray/black), and QueE+FldA (light/dark green). Increases in CDG production due to the presence of 0.5 µM MV are indicated by arrows. Data shown are an average of three experiments. 74 mixture. As expected, in the presence of FldA there is significant enhancement of the production of CDG (0.6 v. 4.7 min-1, ~ 8-fold). In the presence of MV, there is an additional increase in activity to 7.8 min-1, which represents an overall 13-fold increase relative to DT alone. These data further support the notion that rates measured in the ECF system likely represent a biomolecular reaction, whereby Fld on one protein is activating a QueE on a different polypeptide chain. There is an important caveat to consider when interpreting the results shown above. In these experiments, we have made the assumption that access to the QueE is not obstructed in the ECF system. However, no optimization of the interaction has been made in these experiments. Indeed, the placement of FldA at the N-terminus of QueE is somewhat arbitrary. Future studies where the FldA domain is moved, and/or the tether is shortened or lengthened could provide additional insights. We carried out control experiments to evaluate whether FldA in the chimeric protein can be reduced in a similar manner to the FldA monomer, and that the differences in reactivity do not result from differences in the reduction profiles. We initially attempted to reduce FldA, ECF, and ECF∆RS by titrating with various concentrations of DT. While Fld can reduce QueE, albeit at concentrations that exceed stoichiometric DT, ECF and ECF∆RS appear almost completely unaffected by increasing concentrations of DT (see Figure 3.7). This is quite intriguing, as it may suggest that the Fld may be somehow “protected” under these conditions. Interestingly, however, when 5 µM MV is added to the titrations (Figure 3.8), we observe quantitative conversion of the oxidized FMN to the semiquinone, and thence to the fully reduced flavin. The titration profiles at 467 and 580 nm, corresponding to the oxidized and the semiquinone flavin (Figure 3.9), are essentially 75 Figure 3.7 – Spectroscopic Reduction of FldA with DT. UV-Visible changes to the spectra of A) FldA, B) ECF, and C) ECF∆RS were determined at various concentrations of DT. 76 Figure 3.8 – Spectroscopic Reduction of FldA with DT in the Presence of MV. UVVisible changes to the spectra of A) FldA, B) ECF, and C) ECF∆RS were determined at various concentrations of DT in the presence of 5µM MV. In (A,C, E) black is the fully oxidized protein prior to addition of DT (transition to gray). The dotted line indicates full formation of the semiquinone. Dark to light blue lines indicate formation of fully reduced FMN. Isosbestic points are indicated by colored arrows indicating the first electron acceptance (black) or second (blue) event. 77 Figure 3.9 – Spectroscopic Reduction Replot of Oxidized and Semiquinone Features. UV-Visible changes to the spectra of A) FldA, B) ECF, and C) ECF∆RS were determined at various concentrations of DT (gray) or DT+ MV (black). The spectral changes at 467 and 580 nm corresponding to oxidized and semiquinone of the flavin are shown in the absorbance replots. Representative results from two experiments are shown in solid and dotted lines. 78 identical. 3.4. Conclusion A quantitative interpretation of these results is difficult at this time. The titrations include three coupled equilibria: FldoxóFldsq, FldsqóFldred, QueEoxóQueEred. Since MV is required to observe reduction of bound FMN, the measurement is essentially limited by the midpoint potential of the FMN (-124 and -314mV).34 We were initially hoping that we may be observe differences in these profiles that may suggest which form (Fldsq v. Fldred) is involved in reducing the QueE. Future experiments to probe this should utilize mediators of different potentials. 3.5 Materials and Methods 3.5.1. Protein Expression and Purification 3.5.1.1. E. coli QueE-FldA Fusion An N-terminal fusion of the QueE and FldA homologs from E. coli was prepared where the two proteins were connected via a 30 aa linker (Figure 3.10). A hexahistidine tag was included at the N-terminus to permit rapid purification. The chimera was cloned into pet28a and E. coli BL21(DE3) cells were cotransformed with pDB1282 (REF), which encodes the isc operon for [4Fe-4S] cluster biosynthesis. Transformants were plated on LB containing 50 µg/mL kanamycin and 100 µg/mL ampicillin and grown at 37 °C. Starter cultures were prepared from single colonies and used to inoculate 1.8 L flasks containing 1 L LB and both Kan and Amp. The cells were grown at 37 °C to OD600nm~0.3, at which time 0.05% arabinose was added to induce 79 GSSHHHHHHSSGLVPRGSHMAITGIFFGSDTGNTENIAKMIQKQLGKDVADVHDIAKSSKEDL EAYDILLLGIPTWYYGEAQCDWDDFFPTLEEIDFNGKLVALFGCGDQEDYAEYFCDALGTIRDI IEPRGATIVGHWPTAGYHFEASKGLADDDHFVGLAIDEDRQPELTAERVEKWVKQISEELHLD EILNASLEGGGGSGGGGSGGGGSGGGGSGGGGSASMQYPINEMFQTLQGEGYFTGVPAIF IRLQGCPVGCAWCDTKHTWEKLEDREVSLFSILAKTKESDKWGAASSEDLLAVIGRQGYTAR HVVITGGEPCIHDLLPLTDLLEKNGFSCQIETSGTHEVRCTPNTWVTVSPKLNMRGGYEVLSQ ALERANEIKHPVGRVRDIEALDELLATLTDDKPRVIALQPISQKD DATRLCIETCIARNWRLSMQTHKYLNIA Figure 3.10- Protein Sequence of E. coli Chimeric Fusion Protein. Sequences are color coded based on domain: 6xHis tag (black), FldA (orange), linker (purple), and EcQueE (blue). expression of the isc operson from pBD1282. Expression of the chimeric QueE was induced at OD600nm~0.8 with 0.1 mM IPTG. At the time of induction, FeCl3 (50µM final) was also added to ensure complete occupancy of the cluster. The temperature of the flask was reduced to 18 °C at induction, and cells were shaken at 150 rpm for 16 h. Cells were harvested by centrifugation (6,000 xg) and flash-frozen in liquid nitrogen. Expression of the chimeric ∆RS QueE variant was carried out in the same manner. The QueE-FldA fusion (ECF) was purified by placing 35 g of frozen cells in 0.2 L of a lysis buffer containing 50mM KPi pH 7.4, 0.5 M KCl, and 50 mM imidazole with 1 mM PMSF added. The beaker was placed on ice inside an anaerobic chamber (5%H2, 95% N2) and lysed by sonication (amplitude 50% with the cycle 15 sec on, 45 sec off) for a total of 15 min. The lysate was transferred into a centrifuge bottle, removed from the anaerobic chamber, and clarified by centrifugation at 21,000 xg for 45 min. The lysate was returned to the chamber and loaded onto three pre-equilibrated 5 mL His-Trap columns (GE Lifesciences) equilibrated in the lysis buffer. A linear gradient of Buffer B (containing 500mM Imidazole) was used to elute the fusion protein over 10 column volumes. Fractions 80 containing pure protein were identified based on SDS-PAGE, pooled, and solid NH4(SO4)2 was added over 20 min with vigorous stirring to a 1.5 M final concentration. The resulting solution was loaded onto a 5 mL butyl Sepharose column (GE Lifesciences) equilibrated with 50 mM PIPES (pH 7.4) buffer containing 2 mM DTT and 1.5 M ammonium sulfate. A linear elution profile was used to decrease the ammonium sulfate concentration to 0M similarly over 10 column volumes. Fractions containing the protein were identified on the basis of SDS-PAGE, pooled, and desalted into 50 mM PIPES (pH 7.4) containing 0.1 M KCl and 2 mM DTT using gravity column preloaded with Sephadex G-25 resin. The resulting protein was reconstituted in the anaerobic chamber with 6-fold excess FeCl3 (buffered using saturated sodium bicarbonate pH 8.4), Na2S, and FMN for 4 h. Each concentrated solution was slowly added dropwise to protein solution in a 25mL beaker and allowed to equilibrate for 1 min between additions beginning with FeCl3, then Na2S and finally FMN. The resulting mixture was allowed to remain without stirring in the chamber. Following desalting into 50 mM PIPES (pH 7.4) buffer containing 0.1 M KCl and 2mM DTT, the protein was concentrated to a minimal volume using an Amicon Stirred Cell (Model 8050) pressure driven concentrator with a 10kDa MWCO filter and loaded onto a HiPrep 16/60 Sephacryl S300 high-resolution size exclusion column (GE Lifesciences), which had been equilibrated into the desalting buffer. The protein was eluted isocratically with desalting buffer as the mobile phase (0.5 mL/min). Fractions containing the protein were identified by SDS-PAGE and color, pooled, and again concentrated to a minimal volume. Protein was aliquoted and flash frozen in liquid nitrogen. The protein was stored at -80 C until needed. The ∆RS protein was purified and reconstituted as above. 81 3.5.1.2. E. coli QueE Expression and purification of E. coli QueE was carried out as previously described 12 except that following His-Trap step, fractions containing pure protein were desalted into a 0.1 M KPi (pH 7.4) reconstitution buffer containing 0.5 M KCl, 5 mM MgSO4 and 5% glycerol. This mixture was found to be necessary to stabilize the protein and keep it from precipitating after reconstitution. The purified protein was concentrated using an Amicon Stirred Cell (Model 8050) pressure driven concentrator with a 10kDa MWCO filter, aliquots frozen in liquid N2, and stored at -80 °C until needed. 3.5.1.3. E. coli Flavodoxin The FldA protein used in these studies was expressed and purified as described previously.8, 16 3.5.2. Amino Acid Analysis Determination of accurate amino acids content for cofactor quantification was performed by desalting aliquots of purified protein using an illustra NICK column (GE Healthcare), which had been pre-equilibrated in 10 mM NaOH. Concentrations of the eluted protein were determined with Bradford assay (ThermoFisher) using BSA as a standard, before being submitted for amino acid analysis at the Molecular Structure Facility at the University of California (Davis, CA). Based on these analyses, a correction factor of 0.71 and 0.64 were determined for the chimeric E. coli and the EcQueE, respectively, to relate the Bradford assays to the amino acid content. 82 3.5.3. Elemental Iron Content To determine metal ion content, aliquots of purified protein were diluted in 10% (w/v) nitric acid and submitted to the University of Utah Department of Geology for elemental analysis. Expected iron content was calculated using Bradford analysis and the amino acid correction factor described above for each protein. 3.5.4. Sulfide Content Sulfur content of each protein was determined as previously described.35 3.5.5. FMN Content To determine the FMN content of the proteins, serial dilutions of the protein was carried out in 3% TCA (v/v) final concentration to denature and release the cofactor. Precipitated protein was removed by centrifugation at 14,000 xg for 10 min. The released FMN was analyzed by injecting aliquots (30 µL) on a Vanquish UHPLC (Thermo Fisher), which had been interfaced to a Q-Exactive mass spectrometer to obtain in-line mass spectrometric data of all species. Separation of FMN was performed on a Hypersil Gold C-18 column (2.1×150 mm, 1.9 µm particle size) column with a 30 min gradient of 0-10% acetonitrile in 0.1% aqueous TFA. Concentration of FMN in each sample was determined by comparison to commercially available FMN. 83 3.5.6. Synthesis of Substrates SAM and CPH4 were prepared as previously described.8 3.5.7. Steady-State Kinetic Analysis of QueE All assays were conducted in triplicate under anaerobic conditions. Aliquots withdrawn at various times were quenched with TCA to 3% (v/v) final concentration. The assays were all carried out in 50 mM PIPES•NaOH (pH 7.4), containing 10 mM DTT, 2 mM MgSO4, 2 mM SAM, and 2 mM CPH4. QueE was maintained a 1 µM, which was based on the Fe content. Aliquots of quenched reactions were centrifuged at (21,000 xg) for 10 min to remove precipitated protein. Samples (40 µL) were then withdrawn and injected onto a Vanquish UHPLC (Thermo Fisher), which had been interfaced to a Q-Exactive mass spectrometer to obtain in-line mass spectrometric data of all species. The separation of CDG from other hydrophobic products was performed on a Hypersil Gold C-18 column (2.1×150 mm, 1.9 µm particle size) column with a 30 min gradient of 0-10% acetonitrile in 0.1% aqueous TFA. The concentration of CDG produced was determined by comparing EIC peak area that of a standard. All MS measurements were performed in the positive ion mode with a resolution setting of 100,000 and m/z range of 50 to 650. 3.5.8. Spectrophotometric Reduction with Dithionite Spectral titrations were carried out inside the anaerobic chamber using an Agilent 8453 spectrophotometer. All dithionite solutions were prepared fresh on the day of the experiment and quantified as follows. Aliquots of the stock solutions were mixed with 2,6- 84 dichlorophenoindophenol (DCPIP) (MP Biomedicals). The extinction coefficient of the DCPIP at 600 nm (e = 20.7 mM-1 cm-1) was used to determine the concentration of DT. For the titration experiments, protein to be studied was placed in a solution containing 50 mM PIPES•NaOH (pH 7.4), 0.1 M KCl, and 2 mM DTT. Spectra were collected after the addition of each aliquot of DT. 3.6 References 1. Uematsu, T.; Suhadolnik, R. J., Nucleoside antibiotics. VI. 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Summary of Dissertation Previous studies on the electrochemical properties of sulfonium salts and the +2/+1 couple of [4Fe-4S] clusters have demonstrated that a substantial energetic barrier must be overcome to initiate catalysis in the radical SAM superfamily. The goals of this dissertation have been centered around electron movements both into the reductive [4Fe-4S] cluster and the cofactor S-adenosyl-L-methionine (SAM) itself. We approached these topics in two ways. The data presented in Chapter 2 provide a new experimental benchmark for the upper limit reductive potential of SAM (-1.4 V) through direct electrochemical and downstream LC-MS analysis demonstrating that SAM can undergo 1 electron reduction in solution. The Epc determined in this study is significantly more oxidative than previous estimates. The data suggest no discernable preference for which C-S bond undergoes homolysis. These key features of SAM cleavage will be essential for future discussion on the energetics of this expansive class of enzymes that perform radical chemistry. The results presented in Chapter 3 were focused on the characterization of a chimeric fusion of the radical SAM (RS) enzyme QueE to its cognate flavodoxin (Fld). These efforts were centered on determining the effects of tethering a reductive activation domain to a catalytic domain. The results of these experiments were not conclusive since it was not possible to determine if the observed activity arose from reductive activation of QueE by a Fld embedded in its N-terminus, but from cross activation of QueE by a Fld from a different polypeptide chain.