MECHANISTIC AND SPECTROSCOPIC INVESTIGATIONS OF THE RADICAL SAM MATURASES HydE AND HydG FOR [FeFe]-HYDROGENASE by Jeremiah Nathanael Betz A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry MONTANA STATE UNIVERSITY Bozeman, Montana July 2015 ©COPYRIGHT by Jeremiah Nathanael Betz 2015 All Rights Reserved ii DEDICATION This accomplishment and all others big and small are dedicated to my high school prom date, my goat herder, the mother of my four children, my partner in crime, my fellow sojourner in this crazy beautiful life we have created, my best friend, my loving wife, Megan. iii ACKNOWLEDGEMENTS To my scientific mentor, research advisor, and friend, Dr. Joan Broderick. Thank you for demonstrating how a professional at the top of his/her field can balance a professional and personal life. In the Air Force we never get to choose our bosses; I am blessed to have had the opportunity to select you. To my committee Dr. John Peters, Dr. Brian Bothner, Dr. Joshua Obar, and Dr. Eric Shepard who have given their time and many talents to guide me in my academic journey. Thanks to the other members of the MSU chemistry team like Doreen and Robert for going beyond their job descriptions. The Broderick group is a family given how much we fight and laugh. Amanda thanks for always asking why and challenging me to do the same. It has been an honor to work with my protégé and quiet friend, Anna; I bet someday I’ll be asking you for a job. Thanks Kaitlin for the many laughs and doing the dishes I hid on your bench. I know the lab is in good hands with Kim, Krista, and James keeping the research fires burning. Eric, I don’t have an older brother, but if I did, I hope he would be like you. Thanks for your friendship; our professional and personal paths will certainly continue together. Thanks to the US Air Force Academy for sponsoring my study at MSU, in particular, Dr. Don Bird who held me at gunpoint forcing me go to back to school. Thank you to my extended family, my mom and dad (there is finally a Dr. Betz in the family), Pete and Cindy, Jonathan, Natalie, and Elizabeth for your support of Megan, the kids, and myself. Finally, thanks to my wife and my awesome kids Linus, Juliette, Christian, and Scarlett, you are the bidentate coordinated S-adenosyl-L-methionine and site differentiated [4Fe-4S] cluster in my otherwise ordinary complete (βα)8 TIM barrel. iv TABLE OF CONTENTS 1. INTRODUCTION ...........................................................................................................1 Themes in Radical SAM Enzymes ..................................................................................1 Early Characterization and Superfamily Classification ......................................2 Catalytic Reaction Examples ...............................................................................3 Lysine 2,3-Aminomutase (LAM) ............................................................3 Pyruvate Formate Lyase Activating Enzyme (PFL-AE) .........................6 Biotin Synthase (BioB) ............................................................................7 Molybdenum Cofactor Biosynthetic Enzyme (MoaA) ............................9 Architecture and Mechanism of Radical SAM Enzymes ..................................10 Tertiary Structural Similarities ..............................................................12 Conserved Motifs ...................................................................................14 Common Initial Mechanism ..................................................................18 Iron Sulfur Clusters ........................................................................................................19 Simple Iron Sulfur Clusters ...............................................................................19 Functions of Iron Sulfur Clusters .......................................................................21 Biogenesis of Simple Iron Sulfur Clusters.........................................................21 Nitrogenase Active Site Cluster Maturation ......................................................23 Hydrogenase ..................................................................................................................25 [Fe-only]-Hydrogenase ......................................................................................25 [NiFe]-Hydrogenase ..........................................................................................26 [FeFe]-Hydrogenase ..........................................................................................26 Protein Structure and Active Site H-Cluster ..........................................26 Early Maturation Discoveries ...............................................................27 HydF: The Scaffold Protein ...................................................................29 HydG: Carbon Monoxide and Cyanide Synthase .................................30 HydE: Putative DTMA Bridge Synthase ...............................................33 Research Goals and Highlights ......................................................................................35 Determining HydE’s Substrate, Product, and Reaction Mechanism .........................................................................................................36 Defining HydG’s Substrate Binding Pocket and Tyrosine Lyase Mechanism ...............................................................................37 References ......................................................................................................................38 2. GENERAL METHODOLOGY .....................................................................................51 Cell Growth and Overexpression of Proteins ................................................................51 Fernbach Flask 9 L Growths ..............................................................................51 Fermenter 10 L Growths ....................................................................................53 Purification of HydE and HydG ....................................................................................55 Reconstitution of Radical SAM Enzymes .....................................................................56 v TABLE OF CONTENTS - CONTINUED Protein and Iron Quantitation .........................................................................................58 Synthesis and Purification of S-adenosylmethionine (SAM) ........................................58 References ......................................................................................................................60 3. HydE CONSTRUCT DEVELOPMENT .......................................................................61 Background ....................................................................................................................61 Overexpression of HydE ................................................................................................62 Thermotoga maritima HydE ..............................................................................62 Shewanella oneidensis and Clostridium thermocellum HydE ...........................................................................................63 Shewanella oneidensis HydEF Fusion Proteins .................................................66 Clostridium acetobutylicum Revisited ...............................................................67 References ......................................................................................................................69 4. [FeFe]-HYDROGENASE MATURATION: INSIGHTS INTO THE ROLE HYDE PLAYS IN DITHIOMETHYLAMINE BIOSYNTHESIS ..............................................................70 Contribution of Authors and Co-Authors ......................................................................70 Manuscript Information Page ........................................................................................72 Abstract ..........................................................................................................................73 Introduction ....................................................................................................................73 Experimental Procedures ...............................................................................................74 Cloning, Cell Growth, and Expression .............................................................74 Purification and Reconstitution of HydE ..........................................................74 Electron Paramagnetic Resonance and UV-vis Spectroscopic Characterization ..........................................................................74 SAM Cleavage Assays ......................................................................................74 Analysis of Deuterium Incorporation into dAdoH and SAM ...........................................................................................................75 Molecular Docking Experiments ......................................................................75 Construction of Sequence Similarity and Genomic Context Networks .............................................................................................75 Results ...........................................................................................................................75 Spectroscopic Characterization of HydE ..........................................................75 SAM Copurifies with HydE ..............................................................................77 Substrate Derived Deuterium Atom Incorporation into Deoxyadenosine ..........................................................................................77 Deoxyadenosine Production in the Presence of Select Small Molecules ......................................................................................78 vi TABLE OF CONTENTS - CONTINUED Molecular Docking Studies of Putative HydE Substrates and Analogues ..................................................................................78 Radical SAM Sequence Similarity Networks ...................................................78 Genome Context Analysis .................................................................................79 Discussion .....................................................................................................................79 HydE Cluster Characteristics ............................................................................79 Putative HydE Substrates Define the Active Site ..............................................79 Potential Role of SAM in HydE Catalysis ........................................................79 Mechanistic Insights into HydE Catalysis .........................................................80 Concluding Remarks .....................................................................................................82 Author Information ........................................................................................................82 Acknowledgments .........................................................................................................82 Abbreviations .................................................................................................................82 References ......................................................................................................................82 Supporting Information .................................................................................................85 References for Supporting Information .........................................................................92 5. REACTION MECHANISM OF [FeFe]-HYDROGENASE MATURATION ENZYME HydE ................................................................................93 Abstract ..........................................................................................................................93 Introduction ....................................................................................................................93 Materials and Methods ...................................................................................................94 Growth, Purification, and Reconstitution of HydE ............................................94 Preparation of HydE Variants ............................................................................96 Electron Paramagnetic Resonance Spectroscopy ..............................................97 HydE Turnover Assays ......................................................................................98 Production and Detection of Dehydroglycine ....................................................98 Mapping Conserved Residues and Internal Volume of HydE .............................................................................................100 Results ..........................................................................................................................101 HydE R159 Variants Slow SAM Cleavage .....................................................101 HydE Mercaptopyruvate Lyase Activity .........................................................102 Thallium Nuclear Spin Interaction with FeS Cluster Signal ..................................................................................................104 Discussion ....................................................................................................................106 Putative Active Site Residues ..........................................................................106 Mechanism for Lyase Activity .........................................................................108 Key C-terminal Active Site Residues ..............................................................111 Conclusions ..................................................................................................................113 References ....................................................................................................................115 vii TABLE OF CONTENTS - CONTINUED 6. [FeFe]-HYDROGENASE MATURATION ................................................................118 Contribution of Authors and Co-Authors ....................................................................118 Manuscript Information Page ......................................................................................119 Abstract ........................................................................................................................120 Introduction ..................................................................................................................120 Identification of the [FeFe]-Hydrogenase Maturation Machinery ....................................................................................................................121 HydA Expressed Without Maturases Binds the [4Fe-4S] Cubane of the H-Cluster ...............................................................................122 HydF as a Scaffold or Carrier for the 2Fe Subcluster ..................................................123 HydF Iron-Sulfur Cluster States ......................................................................123 HydF Structure .................................................................................................125 HydF Amino Acid Substitution Studies ..........................................................125 GTPase Functionality and Maturase Interactions ............................................126 Radical SAM Chemistry and Diatomic Ligand Synthesis ...........................................126 Tyrosine as the Source of Carbon Monoxide and Cyanide ............................................................................................................126 Iron-Sulfur Cluster States and Diatomic Ligand Biosynthesis .....................................................................................................127 HydE and the Synthesis of the Dithiomethylamine Ligand? .......................................130 An Unknown Substrate and Mechanism ..........................................................130 HydE Structure and Iron-Sulfur Cluster States ................................................130 Conclusions ..................................................................................................................131 Author Information ......................................................................................................131 Acknowledgements ......................................................................................................131 Abbreviations ...............................................................................................................131 References ....................................................................................................................131 7. INVESTIGATING THE ACTIVE SITES OF HydG ..................................................135 Introduction ..................................................................................................................135 Materials and Methods .................................................................................................136 Mutagenesis of Active Site Residues in C. acetobutylicum HydG ......................................................................................136 Growth, Purification, and Reconstitution of HydG .........................................137 EPR of Rapidly Quenched HydG Turnover Samples ......................................138 EPR of Cyanide Treated HydG-NTM .............................................................138 HydG Turnover Assays and Product Quantitation ..........................................139 Results ..........................................................................................................................141 Capturing a Radical Intermediate of the Tyrosine viii TABLE OF CONTENTS - CONTINUED Lyase HydG .....................................................................................................141 Substrate Promiscuity of HydG .......................................................................143 EPR Spectroscopy of HydG-NTM ..................................................................146 Discussion ....................................................................................................................147 The Tyrosine Lyase Active Site .......................................................................147 Characterization of HydG’s Auxiliary Cluster ................................................151 Conclusions ..................................................................................................................152 References ....................................................................................................................154 REFERENCES CITED ....................................................................................................157 APPENDIX A: Supporting Information for Chapter 7 ....................................................181 ix LIST OF TABLES Table Page 1.1. Radical SAM enzymes with published crystal structure solutions ...........................................................................................11 2.1. Media recipe used for protein overexpression in E. coli of radical SAM enzymes and HydF ....................................................52 2.2. Components used for fermenter growths ........................................................53 2.3. S-adenosylmethionine synthesis .....................................................................59 3.1. Protein overexpression constructs tested and/or built .....................................68 4.S1. EPR characterization of as reconstituted and dithionite reduced HydE samples .................................................................92 x LIST OF FIGURES Figure Page 1.1. The site differentiated [4Fe-4S] cluster ...........................................................2 1.2. Overall reactions of select radical SAM enzymes ...........................................4 1.3. Structure of the LAM .......................................................................................5 1.4. Proposed equilibrium driven mechanism for LAM .........................................6 1.5. Structure of MoaA ...........................................................................................9 1.6. Sequence alignment of select radical SAM enzymes ....................................12 1.7. Plots of substrate mass compared to the completeness of the core domain ..................................................................13 1.8. A sequence alignment logo generated using the Pfam 27.0 database ........................................................................................15 1.9. The anSMEcpe’s hydrogen bond ...................................................................16 1.10. The GGE (PFL-AE) motif interacting with methionine moiety on SAM ........................................................................17 1.11. The reductive cleavage of SAM and the generation of the 5’-deoxyadenosyl radical ................................................19 1.12. Simple iron sulfur clusters in biology and selected spectroscopic characteristics .........................................................20 1.13. Complex metallocofactors ...........................................................................23 1.14. Crystal Structure solution for C. pasteurianum [FeFe]-hydrogenase CpI ..............................................................................27 1.15. Proposed composite [FeFe]-hydrogenase maturase scheme ..........................................................................................30 2.1. SDS-PAGE gel of C.acetobutylicum HydE with C-terminal tag ................................................................................................55 xi LIST OF FIGURES – CONTINUED Figure Page 2.2. Protein purification and reconstitution images ..............................................57 3.1. HydE overexpression and purification from Thermotoga maritima, Shewanella oneidensis, and Clostridium thermocellum .......................................................................62 3.2. EPR Spectra of C. thermocellum HydE .........................................................65 3.3. C. acetobutylicum HydE images ....................................................................68 4.1. [FeFe]-hydrogenase crystal structure with H-cluster .....................................74 4.2. UV-vis spectra of C. acetobutylicum HydE as-purified and following chemical reconstitution ........................................76 4.3. Low temperature EPR spectra of C. acetobutylicum HydE .................................................................................76 4.4. Results of analysis for deuterium incorporation ............................................77 4.5. Stimulation of 5’-deoxyadenosine production ...............................................78 4.6. Active site of T. maritima HydE ....................................................................79 4.7. Cartoon network representing the relationship of HydE family ..............................................................................................80 4.8. Abridged genomic context network for HydE enzymes ..........................................................................................................81 4.S1. Structure-based multiple alignment of HydE homologs ......................................................................................................86 4.S2. D-atom incorporation into dAdoH and putative substrate structures ........................................................................87 4.S3. Clostridium acetobutylicum HydE spectra ..................................................88 xii LIST OF FIGURES – CONTINUED Figure Page 4.S4. EPR Spectra of dithionite reduced HydE ....................................................89 4.S5. Larger genomic context network for HydE enzymes ........................................................................................................90 4.S6. Glyoxylate standard titration curve and HydE SAM cleavage assay with cysteine .............................................................91 5.1. UV-vis spectroscopy of pre and post reconstituted HydE ........................................................................................95 5.2. 5’-Deoxyadenosine produced WT HydE and the R159A and R159K variants ...................................................................102 5.3. The proposed reaction mechanism for H-atom abstraction by HydE .....................................................................................103 5.4. HydE production of glyoxylate ....................................................................104 5.5. C. acetobutylicum HydE with addition of Tl+ .............................................106 5.6. Active site anionic binding pocket of T. maritima HydE ........................................................................................108 5.7. In vitro synthesis of H-cluster analogs by condensation of formaldehyde and ammonia on a 2Fe scaffold ...............................................................................................109 5.8. HydE Crystal Structures from PDB ID: 3IIZ and 3CIX ..............................................................................................112 5.9. T. maritima HydE crystal structure PDB ID: 3CIX side and bottom zoomed in views ......................................................113 6.1. X-ray crystal structure of Clostridium pasteurianum I [FeFe]-hydrogenase ............................................................121 6.2. Whole cell extract hydrogenase activation result for the Clostridium acetobutylicum maturases ...................................122 xiii LIST OF FIGURES – CONTINUED Figure Page 6.3. Insertion of the 2Fe subcluster into HydA ...................................................122 6.4. Summary of published HydF and HydA FTIR data ....................................123 6.5. Hypothetical coordination environments for the 2Fe subcluster bound to C. acetobutylicum HydF .......................................124 6.6. Hypothetical maturation scheme for H-cluster biosynthesis ..................................................................................................124 6.7. HydF structure .............................................................................................125 6.8. Hypothetical scheme detailing the proposed roles of the different oligomeric forms of HydF in H-cluster biosynthesis ..............................................................................125 6.9. Sequence- and structure-based alignment of radical SAM enzymes with (βα)8 TIM barrels ............................................126 6.10. Hypothetical scheme detailing HydG’s tyrosine lyase activity in the production of the diatomic ligands ..............................................................................127 6.11. HydG structure-based template ..................................................................128 6.12. Tm HydE Structure ....................................................................................130 7.1. Tyrosine analogs used in HydG turnover assays .........................................140 7.2. EPR spectra during turnover showing the generation of the p-hydroxybenzylic radical by HydG ............................................................................................................142 7.3. Broad field X-band EPR spectrum of HydG under turnover conditions ............................................................................143 7.4. 5-Deoxyadenosine and p-cresol produced by HydG with substrate analogs .......................................................................144 xiv LIST OF FIGURES – CONTINUED Figure Page 7.5. EPR spectra of C. acetobutylicum HydG-NTM treated with cyanide .....................................................................................147 7.6. Crystal Structure of Thermoanaerobacter italicus HydG ...............................................................................................148 7.7. Proposed HydG tyrosine lyase mechanism ..................................................150 S1. Primers used for Gibson assembly mutagenesis of HydG variants S338A, Q360E, and Y91F ............................................182 S2. Gibson assembly cloning components and reaction conditions .......................................................................................183 xv LIST OF ABBREVIATIONS aRNR-AE: anaerobic ribonucleotide reductase activating enzyme ATP: adenosine-5’-triphosphate BioB: biotin synthase BLAST: basic local alignment search tool BtrN: butirosin biosynthetic enzyme Ca: Clostridium acetobuytlicum CBI: 1-cyanobenzy[f]isoindole Ct: Clostridium thermocellum CoA: coenzyme A CoM: coenzyme M Cp: C. pasteurianum CpI: C. pasteurianum I [FeFe]-hydrogenase enzyme CTC: C-terminal cluster ΔCTD: HydG without C-terminal domain CW: continuous wave dAdo•: deoxyadenosyl radical dAdoH: 5’-deoxyadenosine DALI: distance alignment matrix method deoxyHb: deoxyhemoglobin DHG: dehydroglycine DRF: 5-deazariboflavin DTMA: 1,3-dithiomethylamine DTT: dithiothreatol Ec: Escherichia coli E. coli: Escherichia coli ENDOR: electron-nuclear double resonance EPR: electron paramagnetic resonance FeS: iron sulfur FLD: fluorescence detector FPLC: fast protein liquid chromatography FTIR: fourier transform infrared spectroscopy γGC: γ-L-glutamyl-L-cysteine GSH: glutathione GTP: guanosine-5’-triphosphate HbCO: carboxyhemoglobin HC: homocitrate Hcy: homocysteine HEPES: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid HemN: coproporphyrinogen III oxidase enzyme HmdB: [Fe-only]-hydrogenase enzyme HPLC: high pressure liquid chromatography xvi LIST OF ABBREVIATIONS – CONTINUED HydA: [FeFe]-hydrogenase HydA∆EFG: HydA expressed without HydE, HydF, and HydG HydAEFG: HydA expressed with HydE, HydF, and HydG HydF∆EG: HydF expressed without HydE and HydG HydFEG: HydF expressed with HydE and HydG HydE: [FeFe]-hydrogenase maturase E HydF: [FeFe]-hydrogenase maturase F HydG: [FeFe]-hydrogenase maturase G HYSCORE: hyperfine sublevel correlation spectroscopy IPTG: isopropyl β-D-1-thiogalactopyranoside ISC: iron sulfur cluster synthesis enzymes ∆iscR: deletion of the isc repressor gene LAM: lysine 2,3-aminomutase LB: Luria broth LC-MS: liquid chromatography mass spectrometry LipA: lipoyl synthase enzyme βME: β-mercaptoethanol MiaB: tRNA modifying enzyme MoaA: molybdopterin biosynthetic enzyme A MP: mercaptopyruvate 3-MPA: 3-mercaptopropionic acid MTA: methylthioadenosine MWCO: molecular weight cut off NADPH: nicotinamide adenine dinucleotide phosphate NaDT: sodium dithionite NCBI: National Center for Biotechnology Information NIF: nitrogenase FeS cluster assembly enzymes Ni-NTA: nickel nitrilotriacetic acid resin NMR: nuclear magnetic resonance NosL: tryptophan lyase enzyme NRVS: nuclear resonance vibrational spectroscopy NTM: HydG N-terminal mutant protein PDB ID: Protein Data Bank identification code PFL: pyruvate formate lyase PFL-AE: pyruvate formate lyase activating enzyme PhnJ: phosphonate metabolism enzyme PLP: pyridoxal phosphate PMSF: phenylmethylsulfonyl fluoride PylB: methylornithine synthase enzyme RMSD: root-mean-square deviation RSC: radical SAM cluster xvii LIST OF ABBREVIATIONS – CONTINUED QueE: 7-carboxy-7deazaquanine synthase enzyme SAH: S-adenosylhomocysteine SAM: S-adenosylmethioinine SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis SFLD: Structure Function Linkage Database So: Shewanella oneidensis SPL: spore photoproduct lyase enzyme SUF: FeS cluster assembly proteins ThiC: thiamin pyrimidine biosynthetic enzyme ThiH: thiamine biosynthesis enzyme H TIM: triosephosphate isomerase Tm: T. maritima Tn: T. neopolitana Tt: Thermoanaerobacter tengcongensis TWB: thesis writing burnout WT: wild type XAS: X-ray absorption spectroscopy YfiD: radical containing enzyme xviii ABSTRACT While biochemical, spectroscopic, and analytical investigations helped classify multiple phylogenetically distinct hydrogenases it was not until 2004 that Peters et al. gave the world a look at the non-proteinaceous component of the active site of a hydrogenase enzyme. The active site (H-cluster) of [FeFe]-hydrogenase was found to possess a typical [4Fe-4S] cluster bridged by the sulfur of a cysteinyl group to an iron of a uniquely decorated 2Fe subcluster that serves as the site of molecular hydrogen synthesis and oxidation. The subcluster contains two irons bridged by a dithiomethylamine (DTMA) group and a carbon monoxide ligand. In addition each iron is coordinated by a carbon monoxide and cyanide ligand. Posewitz et al. in 2004 were the first to shed light on the syntheses of these non-proteinaceous ligands when through an insertional mutagenesis study of a hydrogen producing green alga they found two radical SAM enzymes, HydE and HydG, that were required for the maturation of [FeFe]- hydrogenase. HydG has been extensively studied and been shown to produce the diatomic ligands of the H-cluster from tyrosine. In this work the substrate specificity and active site of HydG was investigated. These investigations led to a refinement of the location and mechanism of H-atom abstraction of the substrate HydG and support the identity of the C-terminal FeS cluster as a [4Fe-4S] cluster that is responsible in the later steps of diatomic production. While several crystal structures of HydE have been published, the work reported herein is the first to propose a substrate and reaction mechanism for HydE. The results point to commonly biologically available low molecular weight thiols such as L-cysteine, L-homocysteine, and mercaptopyruvate as likely substrates. More recent work has implicated mercaptopyruvate as the substrate given glyoxylate was produced under turnover conditions. Our proposed mechanism involves formation of thioformaldehyde from mercaptopyruvate. Two thioformaldehyde units may be condensed with ammonia forming the DTMA precursor. While many details remain unsolved regarding the maturation of [FeFe]-hydrogenase, our findings regarding HydE and HydG are important steps forward in the understanding of biological catalysts of hydrogen production. 1 CHAPTER 1 INTRODUCTION Themes in Radical SAM Enzymes The abstraction or transfer of a hydrogen atom (H•) from an unactivated bond in vivo is often carried out by transition metal containing enzymes. Due to its multiple biologically accessible oxidation states, iron is more often than not called upon to aid in hydrogen atom abstraction mechanisms. This is illustrated in one major class of heme containing proteins, cytochrome P450 that under anaerobic conditions may utilize an oxyferryl heme group to abstract an H-atom from a C-H bond eventually producing an alcohol.(1) This oxygen insertion reaction is used in solubilizing foreign hydrophobic chemicals and synthesizing biologically important chemicals. The radical S-adenosyl-L- methionine (SAM) superfamily,(2) on the other hand, is primarily responsible for anaerobic H-atom abstraction events that initiate a wide array of biochemical transformations. Radical SAM enzymes all contain a [4Fe-4S] cluster, in which three of the irons are coordinated by sulfurs from three cysteine residues and one by the carboxyl and amine functional groups of SAM in a bidentate fashion (Figure 1.1). Over 5000 anaerobic and aerobic organisms from all three domains of life possess radical SAM enzymes. Roughly fifty thousand radical SAM enzymes have been putatively identified through bioinformatics analyses of sequences in the NCBI sequence database illustrating their importance in biology. 2 Figure 1.1. The site differentiated [4Fe-4S] cluster coordinated by the Cys residues of the CX3CX2C motif and SAM (PDB ID: 3IIZ). Atoms in figures are colored as follows: Green (C), Blue (N), Red (O), Yellow (S), and Orange (Fe) unless otherwise noted. Early Characterization and Superfamily Classification As one can only see the forest when the number of trees is large and the vantage point is sufficiently far, the description of the radical SAM superfamily arose only after the discovery and biochemical and spectroscopic characterization of several key enzymes. New bioinformatics tools and low cost sequencing led to the discovery of many representative enzymes further catalyzing the superfamily designation.(2, 3) The classification scheme is largely built around the highly conserved CX3CX2C motif whose thiolate moieties bind three irons of a site differentiated [4Fe-4S] cluster. Early members, which are still currently used as model enzymes for understanding radical SAM mechanisms, include lysine amino mutase (LAM), pyruvate formate lyase activating enzyme (PFL-AE), and biotin synthase (BioB).(4) While these enzymes 3 catalyze very different overall chemistries it is held that their early mechanistic steps are conserved, as later described. Catalytic Reaction Examples A small sample set of the breadth of reactions that radical SAM enzymes are capable of catalyzing is represented in Figure 1.2: isomerizations (A and E), enzyme activation (B), sulfur insertion (C), amino acid cleavage (D), and complex rearrangements (F). Four examples of some of the best characterized radical SAM enzymes are highlighted in the following sections. Lysine 2,3-aminomutase (LAM) Radical SAM enzymes are often employed to catalyze reactions involving amino acid metabolism. LAM, for example, stereoselectively converts L-lysine to L-β-lysine in the first step of the lysine fermentation pathway. LAM was shown to require SAM and the cofactor pyridoxal phosphate (PLP) for full mutase activity and activity enhanced with the addition of exogenous iron (Fe2+).(5) Subsequent investigations showed that four irons were bound per subunit, and these were assigned to a [4Fe-4S] cluster.(6) Electron-nuclear double resonance (ENDOR) studies demonstrated that SAM coordinated to this [4Fe-4S] cluster via the amino and carboxylate moieties, and suggested that upon reductive cleavage the methionine would be bound as a tridentate chelate with the added coordination of the sulfur.(7) The X-ray crystal structure of LAM from Clostridium subterminale revealed a dimer of dimers where each structural unit contains a splayed (βα)6 motif (Figure 1.3A and B) similar to a TIM (βα)8 barrel.(8) LAM is rather unusual amongst radical SAM 4 enzymes as its quaternary structure is stabilized by both zinc ion binding and domain swapping.(8) Figure 1.2. Overall reactions of select radical SAM enzymes. A) Lysine 2,3-aminomutase (LAM). B) Pyruvate formate lyase-activating enzyme (PFL-AE). C) Biotin synthase (BioB). D) Thiamine biosynthesis enzyme H (ThiH). E) Pyrrolysine biosynthesis enzyme B (PylB). F) Molybdopterin biosynthetic enzyme A (MoaA). R = triphosphate group. 5 Figure 1.3. Structure of the LAM subunit as viewed from side (A) and bottom (B) of the partial TIM barrel. Alpha helices in light blue and beta strands in magenta. The active site highlighting the [4Fe-4S] cluster, SAM, PLP, and Lys (C). The distance in Å between lysine’s carbon with abstractable hydrogen and 5’C of SAM marked with yellow dash (PDB ID: 2A5H). The LAM isomerization reaction (Figure 1.4) is completely reversible, with SAM used as a cofactor and regenerated after each catalytic cycle.(9) PLP forms a Schiff base with the α or β amine nitrogen of lysine immobilizing the substrate. Upon reductive cleavage of SAM, the 5’-deoxyadenosyl radical (dAdo•) intermediate is produced, which abstracts a hydrogen atom from lysine to initiate rearrangement. The active site is located within the partial TIM barrel, and the distance between C5’ of SAM and the β carbon of lysine was determined to be 3.6 Å (Figure 1.3C);(8) at this distance the radical can swing over and abstract a hydrogen from either the β carbon of lysine or the α carbon of β-lysine.(9) The generally accepted isomerization mechanism has a cyclic intermediate that includes the α carbon and nitrogen and the β carbon of lysine (Figure 1.4) in a manner that is likely similar to the analogous enzyme glutamate 2,3-aminomutase.(10, 11) 6 Figure 1.4. Proposed equilibrium driven mechanism for LAM following reductive cleavage of SAM. Pyruvate Formate Lyase Activating Enzyme (PFL-AE) While LAM has a relatively small substrate, PFL-AE abstracts a hydrogen atom from a conserved glycine residue on the enzyme pyruvate formate lyase (PFL), a 170 kDa dimer. The proposed mechanism begins by coupling the oxidation of a [4Fe-4S]1+ cluster to the reductive cleavage of SAM, which produces methionine and the reactive dAdo• radical.(12, 13) PFL- AE induces a large conformational shift in PFL causing it to expose Gly-734.(14) The dAdo• radical then directly abstracts the pro-S hydrogen from Gly-734 of PFL allowing for the generation of a relatively stable radical on PFL (Figure 1.2B).(14-16) Activated PFL can then reversibly catalyze multiple conversions of coenzyme A and acetate to acetyl- CoA and formate. 7 Upon exposure to O2, activated PFL is cleaved into two catalytically inactive peptides with the masses of 3 and 82 kDa.(17) Some organisms produce a “spare part” to repair the inactivated PFL enzyme; in E. coli this spare part is the protein YfiD, which can restore activity after binding to the larger PFL degradation product.(18) YfiD contains a catalytic glycine residue located in a 50 amino acid sequence that corresponds to a similar sequence in PFL. While full activity is observed only with these natural substrates, lower level glycyl radical formation can be observed when as little as a heptamer peptide fragment of the glycyl radical loop is shared by PFL and YfiD.(16) Unlike LAM, PFL-AE employs SAM as a cosubstrate and thus SAM must be continually resupplied for catalytic activity. PFL-AE is the enzyme for which the SAM coordination to the site-differentiated [4Fe-4S] cluster was first demonstrated;(7, 19) this same coordination mode has now been observed in all radical SAM enzymes for which structural information is available, suggesting that the SAM-cluster interaction is an essential aspect of the radical SAM mechanism.(20) Biotin Synthase (BioB) The biologically essential vitamin biotin is synthesized from dethiobiotin in vivo by biotin synthase. This enzyme is the first documented and most studied member of the subfamily of radical SAM enzymes that insert sulfur into unactivated C-H bonds. The disputed role or roles of auxiliary [2Fe-2S] and [4Fe-4S] clusters in sulfur inserting enzymes has led to lively discussions between research groups, with the primary point of contention being whether auxiliary clusters are immediately sacrificed to provide the substrate sulfur, or exogenous sulfur supplies are tapped during or even prior to catalysis. In addition to the canonical [4Fe-4S]2+ cluster observed in all 8 other radical SAM enzymes, biotin synthase was found to contain an auxiliary [2Fe-2S]2+ cluster as demonstrated by Mössbauer spectroscopy with differential isotopic iron labeling.(21) While multiple lines of evidence indicated that SAM was not the sulfur source for biotin biosynthesis,(22, 23) other experiments demonstrated that when 35S- reconstituted BioB was assayed, 35S was inserted into dethiobiotin to produce labeled biotin.(24, 25) Further spectroscopic studies indicated that the [2Fe-2S] cluster was degraded during turnover, suggesting that this cluster served as the sacrificial sulfur donor during biotin synthesis.(26) Such a role for the [2Fe-2S] cluster was also supported by the X-ray crystal structure of BioB, which revealed this cluster optimally positioned to react with the dethiobiotin subsequent to H-atom abstraction.(27) The current mechanistic proposal for BioB involves the cannibalistic abstraction of a sulfide from the [2Fe-2S] cluster by two sequential rounds of radical mediated hydrogen atom abstraction from the methyl and then methylene groups of dethiobiotin to produce an initial 9-mercaptodethiobitin intermediate and then finally biotin.(28) In addition to supplying the sulfur for biotin production, it has been proposed the [2Fe-2S] cluster may also act as a radical quenching Lewis acid after both hydrogen atom abstraction events.(29) The eventual replenishment of the incorporated sulfur in vivo is likely supplied by cysteine desulfurase activity. Some have proposed that as biotin synthase is partially degraded during turnover it should be referred to as a substrate and not an enzyme.(24) The sources of sulfur in other similar sulfur-insertion enzymes such as lipoyl synthase (LipA) and MiaB are still under investigation.(30-32) 9 Molybdenum Cofactor Biosynthetic Enzyme (MoaA) In 1998 investigators shed significant insight into the complex intramolecular rearrangements that yield the precursor to the molybdopterin cofactor (Figure 1.2F).(33) Cells containing a moaABC cassette that were supplied with isotopically labeled ribulose 5-phosphate (pentose) or guanine produced labeled molybdopterin precursors as identified by NMR spectroscopy. The pentose and guanine moieties in GTP have since been confirmed to be the sources of the molybdopterin skeleton.(34, 35) Hänzelmann and Schindelin solved a crystal structure of the MoaA radical SAM enzyme in 2004 and confirmed the presence of an additional [4Fe-4S] cluster located ~17 Å from the canonical N-terminal [4Fe-4S] cluster.(35) The same authors solved another MoaA crystal structure with bound substrate, 5’-GTP, and the SAM degradation products methionine and 5’-deoxyadenosine bound which surprisingly revealed that GTP coordinated to the accessory cluster (Figure 1.5).(34) Figure 1.5. Structure of MoaA as viewed from side (A) and bottom (B). The active site highlighting the two [4Fe-4S] clusters, 5’-deoxyadenosine (dAdo), methionine, and GTP (C) (PDB ID: 2FB3). ENDOR studies carried out in collaboration with Brian Hoffman clarified GTP coordination to the second cluster in an unusual bidentate fashion to the enol tautomer of guanine.(36) With structural information from the active site and deuterium labeled GTP, 10 researchers in 2013 identified the likely hydrogen abstraction site to be the 3’C of the ribose moiety and subsequently proposed a mechanism for GTP rearrangement.(37) Architecture and Mechanism of Radical SAM Enzymes In addition to similarities in the primary structure, radical SAM enzymes share elements of the same tertiary fold.(38) Two years after the formal classification of the superfamily the first crystal structure of a radical SAM enzyme (HemN) was solved.(2, 39) The HemN active site structure was consistent with earlier spectroscopic investigations of PFL-AE that had demonstrated coordination of the amino and carboxy groups of the methionine moiety of SAM to the site differentiated [4Fe-4S] cluster.(7, 19) HemN was also shown to consist of two domains, one of which was a (βα)6 barrel. To date about two dozen other radical SAM crystal structures have been published. Each enzyme is shown to contain a modestly modified TIM barrels, generally either a complete (βα)8 or an incomplete (βα)6 TIM barrel with the exceptions BtrN and QueE.(40, 41) Table 1.1 highlights some of the details of theses structures. In addition to the core TIM barrel motif, many of the solved structures contain smaller auxiliary domains that are believed to aid in substrate recognition and help seal the active site from bulk solvent. The TIM barrel serves as a convenient container for the unique initiating mechanism common to all radical SAM enzymes. 11 Table 1.1. Radical SAM enzymes with published crystal structure solutions. 12 Tertiary Structure Similarities The smallest radical SAM enzyme characterized is the anaerobic ribonucleotide reductase activating enzyme (aRNR-AE), which has been predicted to have a (βα)4 barrel although its structure has not yet been solved.(38) The crystal structures of the radical SAM enzymes, BtrN and QueE, have recently been solved and exhibit significant modifications of the core (βα)6 barrel.(40, 41) BtrN, which catalyzes the anaerobic dehydrogenation of the antibiotic precursor 2-deoxy-scyllo- inosamine, was found to have a unique (β5α4) fold less reminiscent of a typical radical SAM TIM barrel.(38) Nevertheless, like other superfamily members, the CX3CX2C motif was located in a loop between the first β strand and α helix in the primary sequence (Figure 1.6). Figure 1.6. Sequence alignment of select radical SAM enzymes with complete TIM barrels. The CX3CX2C motif that binds the radical SAM cluster is labeled RSC. HydG’s C-terminal domain contains a CX2CX22C motif of which the first two Cys are in a box labeled CTC. 13 7-carboxy-7-deazaguanine synthase (QueE) crystalized as a dimer in which each monomer containing a (β6α3) core.(41) The three helices missing from the core (βα)6 TIM barrel had been largely replaced largely by loops. Alignments of radical SAM enzymes revealed that sequence similarities between different radical SAM subfamilies end around β5 and the differences that extend beyond this region might play a role in substrate diversity.(38, 42) For instance, the completeness of the barrel appears to be inversely correlated with the size of the substrate (Figure 1.7), with the less complete barrels generally accommodating larger substrates (PFL-AE/PFL) and full barrels inherently providing more shielding for radical based chemistry on smaller substrates (BioB/dethiobiotin).(43) Figure 1.7. Plots of substrate mass (Da) compared to the completeness of the core domain. Blue closed circles: Radical SAM enzymes with crystal structures. Red open circles: aRNR-AE (βα)4 and HydG/ThiH (βα)8 predicted completeness. SPL is not plotted because the substrate size is variable, however it would follow the general trend of larger substrates with less complete TIM barrels. Due to the highly modified architectures of QueE and BtrN, they have been plotted as having six and four repeats, respectively. 14 Four exceptions to this overall trend include BtrN, LAM, MoaA, and HemN which all have smaller substrates (< 750 Da) but have only a partial TIM barrel. A stronger correlation occurs with larger substrates, which appear to be completely incompatible with full (βα)8 barrels (Figure 1.7). Conserved Motifs Despite the conserved canonical CX3CX2C motif and a common super-secondary structure, radical SAM enzymes have low sequence homology between subfamilies. However, glycine and proline residues are semiconserved, often punctuating the α helices and β strands. These two residues can relieve stress when transitioning between secondary structural elements by adopting unusual dihedral angles. Structurally based sequence alignments have been helpful in uncovering more motifs by aligning primary structures of radical SAM enzymes in different subgroups (Figure 1.6).(38, 42) These alignments have found two additional semiconserved glycine rich motifs as discussed further below. In 2001 Sofia et al. identified the radical-SAM superfamily, adding over 600 putative members to the five “defining” homologous members.(2) This was accomplished through a bioinformatics approach in which researchers mined the available gene databases for DNA sequences coding for the cysteine rich CX3CX2C motif where X can be any amino acid (Figure 1.8). This motif along with other similarities, such as cofactors and iron and sulfide requirements, had previously been described in the literature but Sofia et al. had not applied these as discriminating factors in the search for other members. Exceptions to the CX3CX2C motif have been reported including in the thiamin pyrimidine biosynthetic enzyme ThiC (CX2CX4C)(44) and in an enzyme involved 15 in phosphonate metabolism, PhnJ (CX2CX27C).(45, 46) Other non-canonical motifs have been described but all contain at least three cysteine residues requisite for ligation of an FeS cluster and subsequent catalysis.(41, 47) For example, while ~20% of QueE sequences contain an additional 11 or 17 residues between the first two cysteines of the three-Cys motif, the insert does not significantly affect the geometry of the cysteines that coordinate the [4Fe-4S] cluster.(41) Figure 1.8. A sequence alignment logo generated using the Pfam 27.0 database. Heights of single letter amino acid abbreviations are proportional to their relative abundances in multiple sequences of radical SAM superfamily members. The three-Cys motif in radical SAM enzymes coordinates a redox active [4Fe-4S] cluster or “radical SAM cluster” whose resting state is 2+; the three-cysteine coordination renders the radical SAM [4Fe-4S] clusters site differentiated, with one of the four irons not coordinated by cysteine. Cuboidal [4Fe-4S] clusters are not unique to radical SAM enzymes and other similarly composed clusters are used in electron shuttling, redox 16 sensing, substrate binding, and protein structure.(48) These [4Fe-4S] clusters typically have biologically accessible reduction potentials. Single electron transporting systems such as flavodoxin, flavodoxin reductase, and NADPH can reduce these clusters to the 1+ state, which is the catalytically active state for radical SAM enzymes.(13) The radical SAM canonical cysteine rich motif is often written CX3CXϕC with ϕ representing an aromatic residue. The graphical representation of the contribution at the penultimate position in Figure 1.8 shows the relative abundance to be F, Y, W, H, and M. Interestingly, the only conserved interaction of this residue is a hydrogen bond (averages distance of 3.0 Å) between the N6 hydrogen of the SAM adenine moiety and the backbone carbonyl on the ϕ residue (Figure 1.9). Figure 1.9. The anSMEcpe’s hydrogen bond between the N6 hydrogen of SAM’s adenine moiety and the backbone carbonyl on the ϕ residue depicted by the yellow dashed line (distance in Å; PDB ID: 4K38). In a recent spectroscopic and functional characterization of BlsE, a radical SAM enzyme required for blasticidin S biosynthesis, which contains a penultimate methionine 17 residue, M37F, M37Y, and M37W mutants had near WT activities.(49) Often radical SAM enzymes without aromatic residues at the ϕ position have such amino acids located within two positions of the C-terminal Cys. The aromatic residue may serve as a conduit for electron transfer to the [4Fe-4S] cluster and/or block solvent access to the active site. A crystal structure of an enzyme with an aliphatic residue would perhaps shed light on the nature of this residue and its role in enzyme function. Figure 1.10. The GGE (PFL-AE) motif interacting with methionine moiety on SAM depicted by yellow dashed lines (distance in Å; PDB ID: 3CB8). The second most prominent radical SAM motif is the conserved GGE sequence starting at the end of the β4 strand. The first Gly of this motif aids in orienting the second Gly and the following Glu residues so that they may form a hydrogen bond with the amino group on the methionine moiety of SAM (Figure 1.10). Further, this “GXIXGXXE” motif is located along the β4 strand, running roughly parallel with SAM and appears to provide the adenosine moiety with a hydrophobic binding pocket. While 18 there are exceptions to these residues in wild type proteins, mutating these residues may lead to inactive proteins.(49) Common Initial Mechanism While the reactions and substrates vary tremendously, radical SAM enzymes appear to initiate catalysis by a common mechanism.(20) Upon in vitro or in vivo reduction to the 1+ state, the radical SAM cluster can donate an electron through an inner sphere electron transfer to the sulfonium moiety on SAM (Figure 1.11). Computational and experimental evidence suggests this electron transfer is promoted by electron donation from the reduced [4Fe-4S] cluster into the σ* orbital of the C-S bond in SAM’s sulfonium moiety.(50, 51) Immediately following the electron transfer event, the S-C(5’) is cleaved to produce methionine and the dAdo•. It is expected that the hydrogen atom to be abstracted would be poised very near to the site where the dAdo• is generated; several crystal structures are in agreement with this expectation. The crystal structure for LAM bound to SAM and the substrate L-α-lysine, for example, shows how the 5’-deoxyadenosyl moiety of SAM is poised in close proximity to the substrate H-atom (Figure 3C). The dAdo• can readily rotate or bend while anchored by the adenosine group to abstract a hydrogen atom from the nearby substrate. The substrate is held in very close proximity to the radical so that very little movement is needed.(52) The substrate-based radicals then embark on a seemingly limitless array of transformations. 19 Figure 1.11. The reductive cleavage of SAM and the generation of the 5’-deoxyadenosyl radical (dAdo•). Iron Sulfur Clusters The simplicity of iron sulfur cluster structures underrepresents the diversity of biological roles to which they have been called to perform. Of the seven most common metals in earth’s crust (aluminum, iron, calcium, sodium, potassium, magnesium, and titanium) iron is unique in its ability to readily adopt multiple oxidations states at or near physiological conditions.(53) Sulfur’s utility and plasticity, on the other hand, does not necessarily stem from its range of oxidation states (2- to 6+) but rather its ability to donate into 3d orbitals forming very covalent bonds with iron. Sulfur, in its acid labile (S2-) and cysteinyl thiolate forms serves as the core for a wide variety of protein cofactors.(48) Simple Iron Sulfur Clusters While iron most often adopts a tetrahedral coordination when bound to sulfur, often period two elements increase iron’s coordination environment to five or six.(54, 55) The most common FeS clusters in biology are [2Fe-2S], [3Fe-4S], and [4Fe-4S] as illustrated in Figure 1.12.(56) All three cluster types can be reduced with biological ferredoxin and flavodoxin reducing systems.(48, 57) In the radical SAM enzymes involved 20 in the maturation of [FeFe]-hydrogenase, all three common FeS cluster types are present either in the active enzyme or as degradation products. Formal charges and valence delocalization properties have been assigned to each iron in the three cluster species through analysis of Mössbauer spectra.(58-60) Most relevant to radical SAM enzymes are the site differentiated [4Fe-4S] clusters.(20) The 2+ oxidation state of the [4Fe-4S] cluster can simply be described as two valence delocalized Fe2.5+-Fe2.5+ pairs that are antiferromagnetically coupled.(54) The reduced [4Fe-4S]1+ cluster can be described as a single mixed valence Fe2.5+-Fe2.5+ pair antiferromagnetically coupled to a Fe2+-Fe2+ pair.(61) Figure 1.12. Simple iron sulfur clusters in biology and selected spectroscopic characteristics. 21 Functions of Iron Sulfur Clusters In proteins, FeS clusters serve four broad interrelated functions: (1) electron transfer, (2) substrate binding/activation, (3) redox sensing, and (4) tertiary structure stabilization.(48, 62) The iron sulfur clusters in the radical SAM enzymes HydE and HydG can be assigned all four of these roles as will be described later in Chapters 4 and 7. Biogenesis of Simple Iron Sulfur Clusters While in vitro reconstitution of iron sulfur clusters can be achieved by reconstitution with free exogenous iron and a source of sulfide in an anaerobic, reducing environment, these conditions are far from physiological and are not achievable inside a cell.(63, 64) Further, this relatively crude method for loading iron and sulfur clusters into proteins is in stark comparison to the three known systems employed by living organisms.(65) The study of the in vivo methods for cluster assembly has been a multidisciplinary effort and the culmination of bioinformatics and biochemical studies have roughly defined three separate roles for the ISC, SUF, and NIF multicomponent systems.(65) The ISC system encoded by the isc operon is generally described as the primary method to generate clusters in vivo. The ISC machinery is largely conserved in prokaryotic organisms and eukaryotic organisms from simple yeasts to human mitochondria.(65, 66) Interestingly, the functionally overlapping and complementary SUF cluster assembly system encoded by the suf operon is usually employed under unfavorable cellular conditions such as limited iron availability, oxidative stress, and the presence of excess heavy metals.(67-69) Both these multicomponent systems share two key features. First, they divide the loading of FeS clusters into apo proteins into an assembly 22 and a transport step. Both possess scaffold proteins upon which a suite of other gene products assemble the clusters. Second, they both have a cysteine desulfurase, which catalyzes the conversion of cysteine to alanine, thereby generating sulfide.(70) In addition to the ISC and SUF multicomponent systems, the NIF system, originally discovered when looking for the iron sulfur assembly necessary for the maturation of nitrogenase, was found in some organisms without nitrogenase.(71, 72) Further investigations will likely reveal more similarities between the systems and further breakdown the canonical definitions as many organisms may have fully functional FeS cluster assembly mechanisms with a patchwork of enzymes from the three multicomponent systems.(73) While simple [2Fe-2S] and [4Fe-4S] clusters can be assembled by two or three generic FeS cluster assembly systems, more complex FeS cofactor assembly requires specific enzymes whose sole function is the construction delivery of unique clusters.(74-77) Figure 1.13 gives four examples of complex iron and sulfur containing cofactors that require their own specific complement of enzymes for construction. 23 Figure 1.13. Complex metallocofactors. (A) [NiFe]-; (B) [FeFe]-; (C) [Fe-only]- hydrogenases; and (D) nitrogenase active site primary coordination environments. Red atoms and bonds represent those putatively derived radical SAM chemistry. Nitrogenase Active Site Cluster Maturation The reduction of elemental nitrogen (N2) to ammonia (NH3) is a costly reaction both biologically and industrially. It has been estimated that two-thirds of all nitrogen fixation is catalyzed by nitrogenase with a bulk of the remainder by the Haber-Bosch process.(78) Diazotrophic microbes catalyze the reaction N2 + 8 H+ 8 e- + 16 MgATP + 16 H2O ! 2 NH3 + H2 + 16 MgADP + 16 Pi at ambient temperatures and pressures whereas industrial processes require much higher temperatures and pressures.(79) The holoenzyme of nitrogenase is composed of two polypeptides, NifD and NifK, with NifD housing the active site. The crystal structure of the NifH(γ)/NifD(α)NifK(β) complex with a (αβγ2)2 stoichiometry from Azotobacter vinelandii likely represents a snapshot of active nitrogenase.(80) The most distinctive aspect of nitrogenase is its active 24 site cluster also known as the M-cluster or FeMo-co (Figure 1.13D) located in NifD. FeMo-co (Fe7MoCS7•homocitrate) contains a unique heterometallic cluster whose core may be described as a [4Fe-3S] cubane bridged to a [Mo3Fe3S] cubane by three sulfides and one hexacoordinated carbide.(81, 82) The molybdenum at the distal position is coordinated in a bidentate fashion to homocitrate (HC) and anchored by a His ligand from the protein. A Cys ligand also from NifD secures the terminal iron on the other side of the cluster. The “ex situ” or external synthesis of the FeMo-co prior to insertion into NifDK is proposed to be accomplished on two discrete proteins.(83) Nitrogenase produced in the absence of the nifB gene was shown to be inactive as it lacked FeMo-co, implicating that NifB was involved in a critical step in cluster maturation.(84-87) The A. vinelandii nifB gene product contains a CX3CX2C motif and a total of nine Cys and eight His residues highly conserved in NifB proteins, indicating the ligation of a site differentiated [4Fe-4S] cluster and potentially other FeS clusters.(84) Anaerobically purified NifB was shown to contain 12 Fe/dimer and could be reconstituted up to 18 Fe/dimer.(87) UV-vis spectral features of reconstituted NifB decrease upon addition of dithionite in a manner suggestive of the presence of FeS clusters. Activity dropped significantly for NifB synthesis of FeMo-co when Fe2+, S2-, or SAM was omitted in in vitro assays. These findings in conjunction with previously noted sequence analysis strongly suggest that NifB is a radical SAM enzyme. NifB likely constructs an all iron FeMo-co precursor that is transferred to the scaffold protein NifEN.(88, 89) Also, NifB has been assigned to the insertion of the central carbon of the FeMo-co precursor using SAM as the substrate.(90) On NifEN, either NifH or NifQ and 25 NifV likely swap the cluster’s distal Fe atom for a Mo atom and aid in its coordination by homocitrate to produce the FeMo-co.(78, 83) This FeMo-co can be transferred to the structural protein complex NifDK where Cys and His residues hold it in place. Hydrogenase Over 70 years ago, Gaffron and Rubin demonstrated that under anaerobic conditions algae could produce molecular hydrogen.(91) They also described how previously hypothesized hydrogenase activity could be stimulated by photoillumination. This opened the door for decades of research in harnessing solar energy for the production of clean fuels such as hydrogen gas. In addition to [FeFe]-hydrogenase, two other phylogenetically distinct classes of hydrogen evolving enzymes have been termed [NiFe]- and [Fe-only]-hydrogenases.(92) [Fe-only]-Hydrogenase [Fe-only]-hydrogenases in methanogenic archaea employ an unusual active site where methenyltetrahydromethanopterin (methenyl-H4MPT+) is catalytically (reversibly) reduced with H2 leading to the production of methane after fixation of CO2.(93) This enzyme harbors a single iron atom which is coordinated by two CO ligands, a cysteinyl residue, and a nitrogen and acyl group from a pyridinol moiety; the moiety has an open ligation site that may interact with H2 (See Figure 1.13C).(94) The maturation machinery for [Fe-only]-hydrogenase is comprised of at least seven separate gene products of which one, HmdB, is a member of the radical SAM superfamily.(95) This maturase enzyme 26 houses a non-canonical CX5CX2C radical-SAM binding motif, however, its exact role in hydrogenase maturation is presently unknown.(96) [NiFe]-Hydrogenase [NiFe]-hydrogenase activity reversibly oxidizes molecular hydrogen to electrons and protons but is usually employed in vivo to catalyze H2 oxidation.(97) The catalytic heart of this subset of hydrogenases contains nickel and iron ions bridged by the thiolate moieties of two Cys residues (see Figure 1.13A). In a similar fashion, two additional Cys residues in a similar fashion further coordinate the redox active Ni atom. The iron ion is also coordinated with two cyanide and one carbon monoxide ligand.(98) The multistep process of inserting metal ions, generating ligands, assembly, and insertion of the cluster into [NiFe]-hydrogenase are accomplished by a suite of six maturase enzymes encoded by the genes hypA-F.(99) None of these six proteins are radical-SAM enzymes and the production of the CO and CN- ligands (diatomics) is not analogous to the method employed by HydG, a radical SAM maturase enzyme of [FeFe]-hydrogenase discussed below. [FeFe]-Hydrogenase Protein Structure and Active Site H-Cluster [FeFe]-hydrogenase (HydA) is primarily used in vivo to catalyze the reduction of protons to hydrogen gas. The first look at [FeFe]-hydrogenase’s multiple domain structure and active site was given by Peters et al. in 1998.(55) A more recent structure better depicts the area of H2 activation composed of a [4Fe-4S] cluster coordinated by four Cys, one of which bridges to a unique 2Fe 27 subcluster. This smaller cluster (see Figure 1.13B) is ornately decorated with several unusual ligands. Figure 1.14. Crystal structure for C. pasteurianum [FeFe]-hydrogenase CpI at 1.39Å. Domain IV contains the H-cluster highlighted in the callout. (PDB ID: 3C8Y). Both iron atoms are singularly coordinated by a carbon monoxide and cyanide ligand and bridged together by as single carbon monoxide as well as two thiolates of 1,3- dithiomethylamine (DTMA).(100-102) During catalysis the [4Fe-4S] cluster may donate and accept electrons to and from the 2Fe subcluster.(103) The in vitro and in vivo activation of this enzyme has been intensely investigated by several research groups whose findings will be highlighted in the following section. Early Maturation Discoveries Ten years ago, 60 years after Gaffron and Rubin’s seminal report on algal hydrogen production, Posewitz et al. determined the maturase enzymes required for [FeFe]-hydrogenase activation.(104) Isolates from a Chlamydomonas reinhardtii insertional mutagenesis library were screened for H2 28 production. A strain unable to produce hydrogen was found to have an insertion in the hydEF-1 gene destroying the activity of the gene product HydEF. HydEF in C. reinhardtii is homologous to two separate proteins (HydE and HydF) found in all prokaryotic organisms that contain [FeFe]-hydrogenase (HydA1 in C. reinhardtii). Genomic analysis and comparison of organisms with [FeFe]-hydrogenases revealed that the gene product of hydG was also strictly conserved. Heterologous co-expression of [FeFe]-hydrogenase, HydEF, and HydG in E. coli was found sufficient and essential to activate HydA1.(104) In the same report the investigators noted that HydE and HydG contain the canonical CX3CX2C motif and are putatively radical-SAM enzymes. Researchers for the past ten years have been uncovering the mechanisms by which [FeFe]-hydrogenase are matured by investigating its maturases individually and collectively. Initial characterization of HydE and HydG demonstrated the requisite spectroscopic and biochemical features that confirmed these two proteins as members of the radical-SAM super family. For example, both were found to contain multiple FeS clusters and reductively cleave SAM.(105) In a hypothesis paper, researchers made several predictions about the roles of the maturation machinery which were later supported experimentally.(106) They speculated that a single enzyme, so as to prevent the build up of one cytotoxic species, would produce both CO and CN-; the maturation scheme would utilize one of the enzymes as a scaffold; and the diatomic ligands may be derived from the components of a glycine radical.(106) Also, they reasoned that the dithiolate bridge would help stabilize a [2Fe-2S] cluster prior to the addition of the diatomic ligands. King, Posewitz, and coworkers followed up their work and demonstrated that C. 29 acetobutylicum maturase genes were more stable in prokaryotic expression systems C. reinhardtii and could activate [FeFe]-hydrogenases from C. acetobutylicum, Clostridium pasteurianum, and C. reinhardtii.(76) This further demonstrated that the maturation systems as well as the H-clusters were highly conserved between multiple organisms and left the door wide open for system level investigations.(107) McGlynn et al. found that inactive HydA lysate could be activated by a lysate containing heterologously co-expressed HydE, HydF, and HydG.(108) They rationalized that their observation of immediate activation upon mixing resulted from one of the enzymes serving as a scaffold for the synthesis of a H-cluster precursor. The same lab later demonstrated that apo-HydA is not capable of activation by a lysate containing coexpressed HydE, HydF, and HydG (HydEFG).(109) Upon reconstitution of HydA a redox active [4Fe-4S] cluster was spectroscopically observed, and the enzyme could then be activated by HydEFG lysate.(108) These results implied that the [FeFe]-hydrogenase maturation machinery was responsible for building and transferring only the embellished 2Fe subcluster to immature HydA. A group out of Stanford University found that addition of L-cysteine and L- tyrosine stimulated the activity of purified C. reinhardtii HydA1 in the presence of dialyzed heterologously co-expressed HydE, HydF, and HydG of Shewanella oneidensis, suggesting possible roles for these amino acids in maturation.(110) Collectively, the research from these groups paved the way for determining the individual duties of the maturation enzymes for [FeFe]-hydrogenase. 30 HydF: The Scaffold Protein While the role of HydA was previously known, the functions of the maturase enzymes where largely speculative. McGlynn et al. furthered their HydF-scaffold hypothesis by demonstrating that isolated and purified HydF, co- expressed in a genetic background with HydE and HydG (HydFEG), could activate [FeFe]-hydrogenase expressed devoid of maturation factors (HydAΔEFG).(108) Following this biochemical result Shepard et al. further spectroscopically characterized purified HydF expressed with (HydFEG) and without (HydFΔEG) HydE and HydG, showing by EPR spectroscopy the presence of both [2Fe-2S]1+ and [4Fe-4S]1+ clusters on HydFΔEG.(111) However, when co-expressed with HydE and HydG, the [2Fe-2S]1+ signal was not observed.(108) Spectroscopy revealed however that HydFEG contained carbon monoxide and cyanide ligands suggesting that the [2Fe-2S] cluster had been converted to the 2Fe subcluster.(108) This expanded on a similar finding from Czech et al. who also observed FTIR Fe-CN and Fe-CO bands when C. acetobutylicum HydF was homologously co-expressed with HydE and HydG.(112) Together these results suggested that the diatomic-ligated 2Fe component of the H-cluster was synthesized on HydF prior to transfer to unactivated HydA. Figure 1.15. Proposed composite [FeFe]-hydrogenase maturase scheme HydF [4Fe-4S] Fe S S Fe HydE SAM HydF [4Fe-4S] Fe S S Fe HydGSAM Tyr NH HydF [4Fe-4S] Fe S S Fe NH NC OC CO OH2 CN CO HydA [4Fe-4S] (Inactive) HydA [4Fe-4S] (Active) Fe S S Fe NH NC OC CO OH2 CN CO HydF [4Fe-4S] 31 HydG: Carbon Monoxide and Cyanide Synthase Despite the limited sequence homology of radical SAM enzymes, which generally pushes the limits of alignment algorithms, HydG was found to share a 27% identity with the thiamine biosynthetic enzyme, ThiH.(113) While tyrosine was shown to be a factor in the synthesis of thiamine in the 1970’s, this requirement was only recently linked to ThiH.(114-116) Under the hypothesis that the similarities between HydG and ThiH may extend to a common substrate, Pilet et al. found that HydG also demonstrated tyrosine lyase activity, producing p-cresol.(113) It was proposed at the time that the dehydroglycine intermediate produced upon cleavage of tyrosine might be a precursor to the DTMA bridge of the H- cluster. Kuchenreuther et al. have recently revisited this hypothesis by investigating HydA activation independent of either HydF (HydE and HydG only) or independent of HydE (HydF and HydG only).(117) Their results led them to speculate that HydG produces the DTMA bridge (in addition to the diatomic ligands discussed below) to form a pre-H-cluster species termed HydG-co; the involvement of HydG in synthesizing the DTMA bridge has not, however, been conclusively demonstrated. The proposed involvement of tyrosine in HydA maturation was further supported by stimulation of H2 production by cell lysates containing heterologously overexpressed HydAEFG upon addition of exogenous tyrosine.(110) Sequence alignments of Thermotoga maritima HydG with T. maritima HydE, Methanosarcina barkeri PylB, and E. coli BioB first indicated that all have (βα)8 complete TIM barrels. This hypothesis was recently confirmed by the solution of two sets of crystal structures by two separate labs.(118, 119) The C-terminal domain beginning 32 immediately after the last alpha helix contains the CX2CX22C believed to take part in the chemistry that differentiates HydG from ThiH.(113) Following the demonstration that tyrosine served as a substrate for HydG, a race ensued amongst several labs to determine the ultimate product(s) and how these fit into the HydA maturation process. In addition to dAdo and p-cresol, HydG was also shown to produce the cyanide ligands likely from a dehydroglycine intermediate.(120) HydG incubated with SAM, Tyr, and NaDT was shown to produce CN−,#which#was#detected via derivatization with naphthalene-2,3-dicarbaldehyde to generate a compound detected by florescence and LC-MS. Utilization of U-[13C,15N]Tyr produced a mass shift in the derivatized product, confirming Tyr as the source of the CN−. Shortly thereafter, it was demonstrated that CO was also produced by HydG.(121) The production of CO was detected by monitoring a shift in the λmax values of deoxyhemoglobin included in the HydG assay as it was converted to carboxyhemoglobin. FTIR analysis of the carboxyhemoglobin produced in the assay carried out with U-[13C,15N]Tyr revealed the expected shifts in the vibrational bands for HbCO, confirming that the CO formed was derived from Tyr. The Swartz lab utilized FTIR analysis to demonstrate that all five diatomic ligands in the H-cluster were derived from tyrosine.(122) Conclusive spectroscopic and analytical evidence that two separate [4Fe-4S] clusters are required for HydG activity was provided in a paper by Shepard et al.(121) Anaerobically expressed, purified, and reconstituted protein was found to contain 8.7 +/- 0.7 Fe/protein and exhibit EPR spectra similar to those previously reported.(105) Upon addition of SAM to the photoreduced and reconstituted HydG, however, a significant 33 shift in the g-values for only one of the two clusters was observed. Further, variants of HydG lacking the C-terminal iron-sulfur cluster, made by either deleting the C-terminal domain (ΔCTD) or by changing the first two cysteine residues of the CX2CX22C motif to serine or alanine, were compromised in their ability to generate the diatomic ligands.(123, 124) While the ΔCTD variant did not produce measurable CO or CN−, the C!S and C!A variants produced half the CN- compared to wild type HydG, with no CO production.(123, 124) Insight into the role of the C-terminal [4Fe-4S] cluster was recently inferred from the observation of Fe(CO)2CN FTIR bands associated with HydG during turnover; this result coupled with ENDOR data tracking 57Fe species suggest that the shuttling of the diatomic species from HydG to HydF may involve cannibalization of HydG’s accessory [4Fe-4S] cluster.(125) A recent crystal structure of HydG was solved with a [5Fe-5S] cluster at the C-terminal terminus; the authors speculate that this may have mechanistic implications for HydG maturation activity.(119) Further discussion pertaining to the mechanisms and substrate binding pocket(s) of HydG will be in Chapter 7. Ultimately, the hypotheses relating to the role of the C-terminal cluster in HydG are very exciting and will certainly incite further investigations. HydE: Putative DTMA Bridge Synthase The same groups that have elucidated the HydF and HydG roles in [FeFe]-hydrogenase maturation have worked on unraveling the function of HydE. An early biochemical characterization of HydE confirmed its place in the radical SAM superfamily by showing that it produced dAdo in the absence of substrate and that it could be reconstituted to contain [4Fe-4S] clusters, but provided little insight into its function or mechanism.(105) The first of several HydE crystal structures 34 confirmed that HydE contains a complete TIM barrel.(126) Crystal soaks and in silico docking studies indicated that the substrate binding site would accommodate a small compound with a carboxylate group and a partial positive charge.(126) Also noted was HydE’s high affinity for SCN- which led to the speculation that HydE produced CN-.(126) More recently, Costantini et al. measured binding constants between HydE, HydF, and HydG by surface plasmon resonance.(127) They determined that HydE and HydG have KD values of 9.19 x 10-8 M and 1.31 x 10-6 M, respectively, for binding to HydF. While neither HydG or HydE could displace each other the high affinity, even under aerobic conditions, further supports the proposed maturation scheme (Figure 1.15) in that HydE operates independently of and prior to HydG in the synthesis of the 2Fe H- cluster moiety. Despite the efforts of several groups, however, the substrate, product, and mechanism of HydE have yet to be determined. HydE has been frequently annotated as biotin synthase (BioB) due to its structural similarities, presence of an auxiliary FeS cluster, and sequence homology to BioB. This has led to proposals that HydE also may have a role in catalyzing a sulfur insertion reaction. However, this proposal is likely inaccurate given that HydE and BioB have significant differences that likely contribute to distinct chemistries. The auxiliary [2Fe- 2S] cluster of BioB is located close to the active site (7.6 Å from the 5’-carbon on SAM) and is likely directly involved in providing the sulfur atom for insertion into dethiobiotin through a 9-mercaptodethiobiotin intermediate.(28) On the other hand, the auxiliary and radical SAM clusters of HydE are located on opposite sides of the TIM barrel at a distance of over 21 Å (PBD ID: 4JY8). At this distance it is unlikely that the sulfur 35 atoms of the auxiliary cluster of HydE could be involved in direct insertion into the substrate in a BioB-like reaction. In addition, the auxiliary cluster in HydE is only semi- conserved and not required for HydA activation.(126) Recently, the crystal structure of methylornithine synthase (PylB) was solved and may provide useful information in the study of HydE.(128) The gene product of pylB is a radical SAM enzyme that catalyzes the isomerization of L-lysine to L-methylornithine in the biosynthetic pathway of pyrrolysine, the 22nd naturally encoded amino acid.(129) The tertiary structure of PylB is an (βα)8 TIM barrel with one conserved [4Fe-4S] cluster coordinated by the canonical CX3CX2C motif. The distance alignment matrix method (DALI) was used to compare PylB to all other solved crystal structures with HydE and BioB scoring 29.0% and 20.6% similarity, respectively.(128, 130) Also, HydE and PylB crystal structures have a root-mean-square deviation (RMSD) of only 1.3 Å compared to BioB and PylB with an RMSD of 1.8 Å. The alignment of PylB and HydE crystal structures show similar SAM and putative substrate binding pockets and together these similarities may point to a parallel mechanism for these two enzymes (Figure 4.7B). Interestingly, HydG and PylB both have proposed mechanisms involving glycine-like intermediates, and perhaps HydE will as well.(123, 128) Work that I performed investigating HydE is captured in Chapters 3-5. Research Goals and Highlights While the maturation of the metallocofactors for [NiFe]-hydrogenase and nitrogenase require several unique maturase proteins, [FeFe]-hydrogenase maturation 36 requires only three specific gene products. Surprisingly, two of these maturases are radical SAM enzymes. The research that was conducted in close collaboration with the hydrogenase team at MSU focused on the chemistries conducted by these two radical SAM enzymes, HydE and HydG. My work on HydE was a continuance from work conducted largely by two graduate students in the Broderick lab, Nick Boswell and Shourjo Ghose. The aims that I originally described in my research proposal were primarily directed toward testing several new hypotheses about HydE. My last year was spent testing a few new hypotheses and the postulations from other groups regarding the chemistry conducted by HydG. Like all scientific proposals, the science more often than not leads you in exciting and unanticipated directions. Determining HydE’s Substrate, Product, and Reaction Mechanism The predominant hypothesis when I began this research was that a hydrogen on the β carbon of cysteine, HydE’s proposed substrate, was the point of abstraction by the deoxyadenosyl radical. This result was suspect for several reasons, one of which was the instability of the enzyme previously used for biochemical and spectroscopic investigations. My first goal was to develop a HydE enzyme system so as not to be limited by enzyme quantity or quality for assay. Secondly, I wanted to further define the role of cysteine in the HydE reaction. While I hypothesized that cysteine had a non- substrate role and proposed a top down LC-MS screening approach for finding HydE’s substrate, I found that the cysteine story was much more interesting than I had previously thought and much of my second and third years were spent elucidating the role of 37 cysteine, culminating in an alternate hypothesis regarding HydE’s substrate and a published manuscript (Chapter 4). Defining HydG’s Substrate Binding Pocket and Tyrosine Lyase Mechanism Our lab in close collaboration with the Peters Lab has an extensive history with the radical SAM enzyme HydG. 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(2014) Crystal Structure of Tryptophan Lyase (NosL): Evidence for Radical Formation at the Amino Group of Tryptophan, Angewandte Chemie. 51 CHAPTER 2 GENERAL METHODOLOGY Cell Growth and Overexpression of Proteins Several FeS-cluster containing proteins were expressed and purified employing the following generalized procedure. While the basic principles were unchanged, the hardware utilized varied depending upon instrument availability and the scale of the overexpression/purification. Details regarding the building and mutation of the specific constructs that were used in the discussed biochemical and spectroscopic assays will be described in Chapters 3, 4, and 7. Following sequence verification, the constructs were transformed into BL21- CodonPlus(DE3)-RIL chemically competent cells which were made using protocols recommended by Agilent Technologies. This cell line contains a plasmid with three tRNA genes that are not commonly found in E. coli but necessary for overexpression of the proteins of interest. This plasmid is maintained by chloramphenicol when required. Transformants were used for pilot expressions and subclones that overexpressed were stored in 30% glycerol at -80 ˚C. Fernbach Flask 9 L Growths In short, phosphate buffered low-salt LB supplemented with iron was used as the growth medium for most of the routine overexpressions of the radical SAM enzymes and HydF and the procedure followed similar previously published methods.(1) Table 2.1 lists 52 the components of the typical recipe for each liter of media. For each 9 L growth the media was divided between six 2.8 L baffled Fernbach flasks, each with 1.475 L, and three 250 mL flasks, each with 50 mL, all of which were then autoclaved. Table 2.1. Media recipe used for protein overexpression in E. coli of radical SAM enzymes and HydF. The night before overexpression, the three 50 mL starter flasks with appropriate antibiotic were inoculated with either a single colony from an LB agar plate or with 1 mm3 of a previously made glycerol stock and placed in a shaker incubator at 37 ˚C and 250 rpm and grown overnight. The next morning the dextrose, appropriate antibiotic, and 7.5 mL of overnight culture were added to each flask and these were incubated at 37 ˚C and 225 rpm for approximately 3 hrs. At an OD600 somewhere between 0.45 to 0.6 the IPTG and half of the ferrous ammonium sulfate were added and the cultures were allowed to incubate at 37 ˚C and 225 rpm for an additional 2.5 hrs at which time they were removed from the shaker incubator and allowed to cool at room temperature for 15 to 30 minutes. The remaining ferrous ammonium sulfate and two drops of antifoam were added to all six flasks. The flasks were then consolidated down to four flasks and the 53 cultures were sparged with nitrogen overnight (14 hrs) at 4 ˚C. The following morning the cells were collected by centrifugation at 6000 rpm for 5 min. The wet cell pellets, which ranged from 16 to 24 grams total, were flash frozen in liquid nitrogen and stored at -80 ˚C until purification. Fermenter 10 L Growths In addition to generating higher cell mass and greater protein yield fermenter growths can also be a simpler choice for protein overexpression with minimal media given the ease associated with adding the trace constituents over the duration of the growth in this method. The minimal media is composed of a mixture of amino acids supplemented with carbon (glucose), phosphate, sulfate, iron, nitrogen (ammonium), calcium, monovalent salts, and vitamins. Component A (Table 2.2) was autoclaved and added to the fermenter the day prior to overexpression. Table 2.2. Components used for fermenter growths 54 Starter cultures were prepared by inoculating 50 mL of LB media and the appropriate antibiotic with a single colony from an LB agar plate or with 1 mm3 of glycerol stock. These overnight cultures were incubated at 37 ˚C and 250 rpm for less than 18 hours prior to inoculation of the minimal media. The day of the growth the fermenter, with Component A, was warmed to 37 ˚C using a thermostatically controlled jacket, agitated at 300 rpm, and then bubbled with filtered compressed air at 5 L/min. During equilibration Component B was made, sterile filtered, and added to Component A in the fermenter. Component C was also added as a dry powders. The entire 50 mL overnight starter culture was added and the OD600 was monitored. At an OD600 between 0.45 and 0.6 IPTG was added for a final concentration of 0.5 mM IPTG and the growth allowed to incubate. After 2 hrs the temperature was actively cooled to 30 ˚C as the compressed air was removed and nitrogen sparging began. At 20 ˚C five drops of antifoam and an additional 0.75 g of ferrous ammonium sulfate hexahydrate were added to the fermenter and then it was placed in a 4 ˚C refrigerator and sparged with nitrogen overnight (14 hrs). The following day the cells were centrifuged at 6000 rpm and the resulting wet cell paste was flash frozen in liquid nitrogen and stored at -80 ˚C until purification. Pre and post expression along with purification samples were taken throughout this process and eventually run on SDS-PAGE gels that were later stained with Coomassie dye to check overexpression levels and relative protein purity (Figure 2.1). 55 Figure 2.1. SDS-PAGE gel of C.acetobutylicum HydE with C-terminal tag. A. Whole cell lysate. B. Precipitated insoluble cell debris. C. Soluble clarified lysate. D. Ni-NTA column flowthrough. E. 10mM imidazole column wash flowthrough. F. 25mM imidazole column wash flowthrough. G. Biorad broad range molecular weight standards. H-M. Single column volume elutions 1-6. Purification of HydE and HydG All subsequent preparation of protein for assays was conducted in gas tight containers or in anaerobic Coy chambers with a 5% hydrogen atmosphere balanced with nitrogen. Previous members of the Broderick Lab have purified heterologously overexpressed polyhistidine tagged HydE and HydG from C. acetobutylicum in a variety of buffers.(2, 3) However, due to protein stability issues, activity optimization, and convenience a single common buffer (CaHyd) composed of 25 mM HEPES pH 8.0, 500 mM KCl, and 5% glycerol (w/v) was found to be optimal for most situations (Chapter 3). Cell lysis was accomplished using a method similar to the one previously described(1) where cell pellets containing overexpressed protein were thawed and incubated in a lysis buffer with a 1:2 cells/buffer ratio. The chemical lysis buffer was composed of CaHyd 56 buffer with 10 mM imidazole and 1% Triton X-100 and supplemented with 9 mg lysozyme, 9 mg PMSF, 1 mg DNAse, 1 mg RNAse, and 100 mg MgCl2 per 50 mL. The cell lysis slurry was stirred vigorously for 2-3 hrs in a strictly anaerobic Coy chamber at 4 ˚C. A clarified lysate was generated by centrifugation at 18K rpm for 30 min at 4 ˚C in sealed Oakridge tubes. For HydE, the clarified lysate was passed over 9 mL of Qiagen Ni-NTA resin that had been equilibrated in lysis buffer (10 mM imidazole CaHyd buffer). When loaded with HydE, the column turned visibly dark brown (Figures 2.1 and 2.2A). The protein bound column was then washed with 7-10 column volumes of lysis buffer and a 25 mM imidazole wash buffer. Finally, the protein was eluted with 250 mM imidazole buffer (figure 2.2B). Colored fractions that ranged from 35 to 330 µM protein were immediately buffer exchanges or dialyzed at 4°C to remove imidazole. This method routinely generated protein that was >95% pure as judged by SDS-PAGE gel with Coomassie staining (figure 2.1). Following removal of imidazole the protein was aliquoted into screw top vials with a rubber o-ring cap, flash frozen in liquid nitrogen, and stored at -80 ˚C. Reconstitution of Radical SAM Enzymes As both HydE and HydG are purified with substoichiometric (< 8 Fe/protein) iron, reconstitution with exogenous iron and sulfide is necessary to achieve fully loaded and active enzymes. The following method, similar to those previously published by our lab(4), can be easily scaled to accommodate any quantity of protein and consistently yields 57 protein with near full loading of iron as judged by EPR and iron quantitation assays (see following section). Figure 2.2. Protein purification and reconstitution images. A. Ni-NTA poly-His affinity column loaded with C. acetobutylicum HydE. B. HydE eluted with imidazole from column. Fractions 1 through 4. C. Reconstitution of 100 µM HydE. D. Sephadex G25 resin in gravity flow column used to exchange buffer and remove imidazole. E. Reconstituted HydE protein concentrated to 344 µM. Pooled HydE fractions were adjusted to a 100 µM final protein concentration and DTT was added to a final concentration of 5 mM prior to reconstitution with exogenous Fe3+ and S2- at 4°C. Over 30 min, six successive aliquots of a 50 mM S2- in water (final concentration of 600 µM/6x protein) were added to HydE. Following the sulfide, 10 mM FeCl3 was added in six additions over 30 min to a final concentration of 600 µM. The solution turned noticeably darker upon each addition of the iron solution (Figure 2.2C). After 2 hrs of incubation with stirring the solution was loaded into an Oakridge gas tight 58 tube and centrifuged for 30 min to remove FeS particulates. The supernatant containing protein was decanted into Amicon 15mL 10 kDa MWCO spin concentrators and spun at 6000 rpm down to ~0.5 mM prior to being buffer exchanged on a 75 mL Sephadex G25 gravity flow column (Figure 2.2D) to remove any remaining DTT, S2-, and Fe2+. The protein (Figure 2.2E) was then aliquoted and flash frozen prior to storage at -80°C in screw top tubes. Protein and Iron Quantitation Protein was quantified using the Bradford method with a BSA standard solution.(5) A colorimetric assay using the spectrophotometric reagent ferrozine was used to quantify the iron content of the protein samples in a manner similar to a previously published method.(6) Synthesis and Purification of S-adenosylmethionine (SAM) SAM was enzymatically prepared in vitro using a crude SAM synthetase lysate similarly to the previously described method.(7) The components listed in Table 2.3A were added to a scintillation vial and incubated overnight (15-16 hrs) with stirring. The reaction was quenched with 1 mL of 1 M HCl causing a noticeable cloudiness due to protein precipitation. The precipitate was removed by centrifugation at 18K rpm at 4 ˚C for 30 min and the supernatant loaded on equilibrated Source 15S cation exchange resin in a GE, HR 10/10 column. SAM binds well to the column given its positive charge and after washing with water and 0.1 M HCl, applying a linear gradient (Table 2.3B) up to 1 59 M HCl was sufficient to elute the SAM. Depending upon the quantity of SAM loaded on the column, the SAM eluted between 0.35 and 0.55 M HCl, which could be easily observed by monitoring the eluent’s absorbance at 260 nm. The eluate containing SAM was concentrated using a rotary evaporator and subsequently lyophilized overnight to a powder. The white to light yellow powder was anaerobically resuspended in 50 mM Tris pH 8.0 buffer and the pH of the solution was then adjusted to 7.2 in an MBraun anaerobic chamber. The concentration of SAM was measured using the extinction coefficient of 16000 M-1 cm-1 and adjusted to 50 mM before aliquoting and storing at -80 ˚C. Table 2.3. S-adenosylmethionine synthesis. A. Components of one pot synthesis of SAM. B. SAM chromatography scheme with cation exchange column. 60 References 1. Shepard, E. M., McGlynn, S. E., Bueling, A. L., Grady-Smith, C. S., George, S. J., Winslow, M. A., Cramer, S. P., Peters, J. W., and Broderick, J. B. (2010) Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold, Proceedings of the National Academy of Sciences of the United States of America 107, 10448-10453. 2. Duffus, B. R., Ghose, S., Peters, J. W., and Broderick, J. B. (2014) Reversible H Atom Abstraction Catalyzed by the Radical S-Adenosylmethionine Enzyme HydG, Journal of the American Chemical Society. 3. Betz, J. N., Boswell, N. W., Fugate, C. J., Holliday, G. L., Akiva, E., Scott, A. G., Babbitt, P. C., Peters, J. W., Shepard, E. M., and Broderick, J. B. (2015) [FeFe]- Hydrogenase Maturation: Insights into the Role HydE Plays in Dithiomethylamine Biosynthesis, Biochemistry 54, 1807-1818. 4. Shepard, E. M., Duffus, B. R., George, S. J., McGlynn, S. E., Challand, M. R., Swanson, K. D., Roach, P. L., Cramer, S. P., Peters, J. W., and Broderick, J. B. (2010) [FeFe]-Hydrogenase Maturation: HydG-Catalyzed Synthesis of Carbon Monoxide, Journal of the American Chemical Society 132, 9247-9249. 5. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72, 248-254. 6. Fish, W. W. (1988) Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples, Methods Enzymol. 158, 357-364. 7. Walsby, C. J., Ortillo, D., Broderick, W. E., Broderick, J. B., and Hoffman, B. M. (2002) An anchoring role for FeS clusters: chelation of the amino acid moiety of S-adenosylmethionine to the unique iron site of the [4Fe-4S] cluster of pyruvate formate-lyase activating enzyme, Journal of the American Chemical Society 124, 11270-11271. 61 CHAPTER 3 HydE CONSTRUCT DEVELOPMENT Background The desire for a hydE construct that overexpresses well, is stable during purification, and amenable to assay with the other maturase enzyme and HydA has led to testing five different constructs and the development of three. Ultimately, a construct with a C-terminal poly-His tag from Clostridium acetobutylicum heterologously expressed in an E. coli strain proved to be the best all around construct for pertinent spectroscopic work and biochemical assays. The following will provide a brief description of the process that led to the selection of this expression system and the recent exploits with this protein. Previous cloning work from the Peters group led to the development of Thermotoga maritima and C. acetobutylicum hydE constructs.(1) However, N. Boswell’s work with C. acetobutylicum HydE was met with difficulty as the ensuing overexpressions resulted in a low yield of protein and the protein often precipitated during purification or upon subsequent handling. Further, pilot expressions and 9 L batch growths from his glycerol stocks did not yield noticeable overexpression as observed by SDS-PAGE (not shown). 62 Figure 3.1. HydE overexpression and purification from Thermotoga maritima, Shewanella oneidensis, and Clostridium thermocellum. Samples’ contents visualized on 10% SDS-PAGE gel with coomassie staining. Overexpression of HydE Thermotoga maritima HydE Even though the T. maritima maturase system has not yet been successfully used to activate HydA, T. maritima HydE’s strong overexpression and thermal stability made it a good candidate for an individual screening assay (Figure 3.1A). T. maritima HydE was grown in 9 L batches in low salt Miller LB media under conditions developed in a collaboration between the Peters and King labs for C. acetobutylicum maturase proteins.(1) Cultures were initially grown under aerobic conditions followed by sparging with nitrogen overnight to aid in cluster assembly and folding of anaerobic proteins. The cell lysate was then anaerobically purified by FPLC yielding a modest 2 mg T. maritima 63 HydE per liter of cell culture. Mass spectroscopic analysis calculated the mass of the purified T. maritima HydE to be 41966.4 amu, which is in close agreement with the predicted transcript. Upon subsequent dialysis 40% of the protein precipitated as quantified by the Bradford method. During this time Clostridium thermocellum and Shewanella oneidensis constructs were developed and attention was diverted to characterize these more promising proteins. Performing a BLAST search using T. maritima hydE yields hundreds of homologous proteins. Among these, S. oneidensis and C. thermocellum were chosen due to accessibility to their genomic DNA for cloning, among other reasons. In fact, the S. oneidensis [FeFe]-maturase system had been successfully used by a competitor’s lab.(2) By using a defined “in vitro” maturase system this lab reported that exogenous tyrosine and cysteine stimulate HydA activity. While most of their work involves using exclusively cell lysates, their expression system has been demonstrated to activate [FeFe]-hydrogenase. HydE from C. thermocellum was also selected because of its origin from a thermophilic organism and, therefore, its potential to exhibit thermal stability. In addition C. thermocellum and the previously studied C. acetobutylicum hydE gene products share a 43.7% identity. Shewanella oneidensis and Clostridium thermocellum HydE S. oneidensis and C. thermocellum hydE genes were each cloned from genomic DNA into a pET14b vector and sequence verified prior to transformation into E. coli expression strains. Pilot growths (See Figure 3.1B) for both constructs were positive for 64 overexpression as was determined by SDS-PAGE of the total cellular protein. While S. oneidensis HydE overexpressed well the protein was likely unstable, folded in a manner that shielded the poly-His affinity tag, or was packaged into inclusion bodies in vivo. This was evident upon chemical lysis of the cell pellet and subsequent FPLC purification of the recovered S. oneidensis HydE fractions that were nearly devoid of FeS clusters and of very low concentration as determined by absorbance at 426 nm and SDS-PAGE, respectively. Further optimization and characterization of S. oneidensis HydE was suspended as concurrent work on the C. thermocellum HydE construct proved to be more promising. As previous HydE constructs often precipitated during the slow loading and washing processes during FPLC, a gravity flow column packed with Ni-NTA resin was used to purify the clarified lysate in order to shorten the purification time. Upon elution with imidazole a recovery of 12.5 mg C. thermocellum HydE per liter of cell culture was achieved (See Figure 3.1C). The protein was found to have a mass of 42259.7 amu that corresponded well with the predicted 42259.9 amu of the holoprotein after its cleavage of the N-terminal methionine. A second large peak that could not be attributed to potassium adduct(s) was also observed in the deconvoluted mass spectrum at 42435.1 amu (175.4 amu higher). This peak may have been due to the presence of a persistent [2Fe-2S] cluster which, according to the literature, is consistent with a 175.8 amu mass found in ESI mass spectra of intact [2Fe-2S] clusters.(3) During handling C. thermocellum HydE, especially during thawing, up to 40% of the protein would often precipitate as was determined by Bradford assays. In a series of 65 freeze/thaw experiments it was found that rapid thawing resulted in a more stable protein and only 10% of the total protein was subsequently lost. In compliance with this observation, upon removal from the -80 °C freezer the anaerobically sealed screw top tube with protein was consistently thawed in a 42 °C water bath prior to entry into the anaerobic chamber. The protein was then reconstituted with Fe2+ and S2- under reducing conditions to attain up to 6.41 iron atoms per protein. The g-values calculated and the temperature relaxation profile from EPR spectroscopy of the purified C. thermocellum HydE indicated the presence of a mixture of [4Fe-4S], [3Fe-4S], and [2Fe-2S] clusters. The complicated spectra that contained shoulders on the pertinent peaks may have been caused by substoichiometric occupation of both [4Fe-4S] clusters (See Figure 7A and 7B). Figure 3.2. A. EPR Spectra of C. thermocellum HydE (100 µM) with 2.4 Fe/protein reconstituted, photoreduced, and photoreduced with exogenous SAM (2 mM); B. EPR temperature relaxation profile. The EPR spectra parameters were microwave frequency 9.37 GHz; temperature 12 +/- 2K unless otherwise noted; power, 1 mW; time constant and conversion time, 20.48 ms; receiver gain, 1 x 104; modulation frequency, 100 kHz; and modulation amplitude, 10 G; C. Plots of production of 5’-deoxyadenosine with time for C. thermocellum HydE (20 µM), SAM (2 mM), and sodium dithionite (1 mM) with and without L-cysteine (200 µM) at 50 °C. 66 While all kinetic assays with C. thermocellum HydE were run at 50 °C (See Figure 2.1C), this was not compatible with C. acetobutylicum HydF because it is unstable at this temperature. This presented a problem in the continuation of S. Ghose’s research. Based on his data, he suggested that in the presence of C. acetobutylicum HydF, C. acetobutylicum HydE produced less 5’-deoxyadenosine (dAdo) and abstracted a proton from the C3 position of cysteine. In order to continue S. Ghose’s research and confirm his results it was necessary to either develop a C. thermocellum hydF construct or reclone C. acetobutylicum hydE in a different vector or coexpress HydE fused to HydF, which has been a successful technique with other similar radical SAM enzymes.(4) Shewanella oneidensis HydEF Fusion Proteins To create a fusion of HydE and HydF I chose to take advantage of the S. oneidensis hyd operon in which hydE and hydF are syntenous. More specifically, the genes hydE and hydF are naturally separated by only 40 nucleotides. The two genes and the noncoding linker were amplified using two sets of primers (Table 3.1) in order to make HydEF fusion proteins that were tagged both N- and C-terminally. The vectors pET-14b and pET-23b were used to make the HydEF fusion with N-terminal and C- terminal 6x His tagged peptides, respectively. To make a single fused protein the stop codon from HydE had to be removed and the proteins had to be put in a single frame. By removing a single adenosine from the TAA stop codon with mutagenesis both requirements were fulfilled. All together four constructs were generated with S. oneidensis hydE and hydF. Table 3.1 provides information regarding the details of the primers, restriction sites, and host plasmids used for the cloning work I performed while 67 conducting my research. The constructs overexpressed well in E. coli BL21(DE3) producing brown proteins with the appropriate masses of 86 kDa. The protein, however, was susceptible to precipitation upon concentration and handling and not extensively assayed. While these proteins showed promise and should be revisited in the future, the C. acetobutylicum HydE protein described below also began to show encouraging results and so investigations into the HydEF fusion proteins were discontinued. Clostridium acetobutylicum Revisited In reviewing radical-SAM and protein purification literature two observations were made. Many radical SAM enzymes such as HydE have the site-differentiated [4Fe- 4S] cluster near the N-terminus. Placing a poly-His affinity tag at the C-terminal end of the protein has generally been found to result in more stable protein.(5) Secondly, purifying and assaying SAM proteins in a buffer with a pH that is more than one pH unit from the protein’s pI is ideal.(6) Perhaps the instability and difficulty with C. acetobutylicum HydE experienced by S. Ghose and N. Boswell was caused by a C- terminal poly-His tag and/or the choice of buffer pH. Indeed, the C-terminal tagged C. acetobutylicum HydE protein (pI = 6.87) is stable during purification, dialysis, reconstitution, spin and sponge well concentration, and multiple freeze/thaw events in a pH 8.0 buffer and overexpresses at adequate levels (See Figure 3.3A and B). Off the column ~3 mg of C. acetobutylicum HydE was recovered per liter of culture. As isolated protein contained 3.7 Fe/protein and could be reconstituted to consistently contain up to 8 Fe/protein (See Figure 3.3C) as determined by colorimetric ferrozine and Bradford assays and confirmed by spectroscopic analysis. 68 Figure 3.3. C. acetobutylicum HydE images. A. Purification HydE elutions; B. SDS- PAGE elution #3; C. UV-vis of pre (black trace) and post-reconstituted C. acetobutylicum HydE (red trace) (83 µM). Table 3.1. Protein overexpression constructs tested and/or built. 69 References 1. McGlynn, S. E., Shepard, E. M., Winslow, M. A., Naumov, A. V., Duschene, K. S., Posewitz, M. C., Broderick, W. E., Broderick, J. B., and Peters, J. W. (2008) HydF as a scaffold protein in [FeFe] hydrogenase H-cluster biosynthesis, FEBS Lett. 582, 2183-2187. 2. Kuchenreuther, J. M., Stapleton, J. A., and Swartz, J. R. (2009) Tyrosine, cysteine, and S-adenosyl methionine stimulate in vitro [FeFe] hydrogenase activation, PLoS.One. 4, e7565. 3. Petillot, Y., Golinelli, M. P., Forest, E., and Meyer, J. (1995) Electrospray- ionization mass spectrometry of molecular variants of a [2Fe-2S] ferredoxin, Biochemical and biophysical research communications 210, 686-694. 4. Lippard, S. J., and Berg, J. M. (1994) Principles of bioinorganic chemistry, University Science Books, Mill Valley, Calif. 5. Lanz, N., Grove, T. L., Gogonea, C., Lee, K., Krebs, C., and Booker, S. J. (2012) RlmN and AtsB as Models for the Overproduction and Characterization of Radical SAM Proteins, Methods in Enzymology 516, 125-152. 6. Kumar, A., and Awasthi, A. (2009) Bioseparation Engineering, I. K. International Pvt Ltd. 70 CHAPTER 4 [FeFe]-HYDROGENASE MATURATION: INSIGHTS INTO THE ROLE HYDE PLAYS IN DITHIOMETHYLAMINE BIOSYNTHESIS Contribution of Authors and Co-Authors Manuscripts in Chapters 4 Author: Jeremiah N. Betz Contributions: Conducted bulk of experimental work, and preparation of most figures and text of the manuscript. Co-Author: Nicholas W. Boswell Contributions: Conducted preliminary assays that contributed to the hypotheses. Co-Author: Corey J. Fugate Contributions: Conducted molecular docking experiments, suggested alternate experiments, and helped interpret results. Co-Author: Gemma L. Holliday Contributions: Prepared sequence similarity network, prepared selected figures and parts of the text of the manuscript, and interpreted results. Co-Author: Eyal Akiva Contributions: Prepared genomic context network, prepared selected figures and parts of the text of the manuscript, and interpreted results. Co-Author: Anna G. Scott Contributions: Prepared protein and reagents and conducted assays. 71 Contribution of Authors and Co-Authors - Continued Co-Author: Patricia C. Babbitt Contributions: Provided important insight and overview for sequence similarity and genomic context networks. Aided in interpretation of results and preparation of the manuscript and figures. Co-Author: John W. Peters Contributions: Provided important insight and overview for biochemical assays and hydrogenase maturation context. Aided in interpretation of results and preparation of the manuscript and figures. Co-Author: Eric M. Shepard Contributions: Conducted preliminary investigations with HydE. Provided preliminary insight and overview for biochemical assays and hydrogenase maturation context. Aided in preliminary interpretation of results and preparation of the preliminary manuscript and figures. Co-Author: Joan B. Broderick Contributions: Provided key insight and overview for entire research program. Facilitated interpretation of results and preparation of the manuscript and figures. 72 Manuscript Information Page Authors: Jeremiah N. Betz, Nicholas W. Boswell, Corey J. Fugate, Gemma L. Holliday, Eyal Akiva, Anna G. Scott, Patricia C. Babbitt, John W. Peters, Eric M. Shepard, and Joan B. Broderick Journal: Biochemistry Status of Manuscript: ____ Prepared for submission to a peer-reviewed journal ____ Officially submitted to a peer-review journal ____ Accepted by a peer-reviewed journal _X__ Published in a peer-reviewed journal Publisher: American Chemical Society Issue in which manuscript appears: Volume 54, 1807-1818 (2015) 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 CHAPTER 5 REACTION MECHANISM OF [FeFe]-HYDROGENASE MATURATION ENZYME HydE Abstract The three unique gene products HydE, HydF, and HydG are necessary for the maturation of the active site H-cluster of [FeFe]-hydrogenase. HydE and HydG are radical SAM enzymes that catalyze chemical transformations by H-atom abstraction and synthesize the nonproteinaceous ligands of the H-cluster. While HydG supplies the diatomic ligands, HydE likely generates the dithiomethylamine (DTMA) group that bridges the 2Fe subcluster at the heart of the H-cluster. A recent multidisciplinary study led to the proposal that HydE uses low molecular mass thiols to generate two units of thioformaldehyde that condense with ammonia producing the DTMA bridge.(1) Here we present our findings of mercaptopyruvate lyase activity by HydE under turnover conditions and expand our hypothesis to include the key residues in turnover and synthesis of the DTMA fragment in the lower part of the TIM barrel of HydE. Introduction While it was recognized early that hydrogen containing molecules were at the core of energy cycling in ecosystems, it was not until 1931 that it was shown molecular hydrogen could serve as an energy source for select bacterial isolates and the term hydrogenase was officially coined.(2) The following several decades of research revealed 94 a tremendous diversity among hydrogenases including oxygen-sensitivity, intracellular location, metallic content, and electron transfer partners.(3) While biochemical, spectroscopic, and analytical investigations helped classify hydrogenases into roughly three groups, termed [FeFe]-, [NiFe]-, and [Fe]-only hydrogenase, it was not until 1998 that Peters et al. gave the world a look at the non-proteinaceous component of the active site of a hydrogenase enzyme.(4) The exact stoichiometry and identity of the unusual ligands of the active site of [FeFe]-hydrogenase, termed the H-cluster, was deciphered over the next thirteen years by several research groups.(5-7) In total, the H-cluster contains a [4Fe-4S] cluster coordinated by four cysteines, one of which bridges a uniquely decorated 2Fe subcluster. The DTMA bridge, and a single carbon monoxide bridge the two irons. Additionally, both irons are coordinated by a carbon monoxide and cyanide ligand. Recently we proposed the DTMA bridge was synthesized from two thioformaldehyde fragments produced by HydE lyase activity on a low molecular weight thiol, perhaps cysteine or mercaptopyruvate.(1) Our current work supports this hypothesis given our observed HydE mercaptopyruvate lyase activity. Other results indicate that the synthesis of the DTMA bridge may occur in a second area of HydE in a stepwise fashion prior to delivery to HydF. Materials and Methods Growth, Purification, and Reconstitution of HydE A detailed description of the cloning including the primers used to build the pET23b-C.a.HydE construct used to express Clostridium acetobutylicum HydE can be 95 found in Chapter 3. The overexpression of the C-terminally 6x-His tagged protein has been described in Chapters 2 and 4.(1) The HydE protein was purified using immobilized metal ion affinity chromatography by a method described in Chapters 2 and 4 and yielded brown colored protein judged >95% pure by SDS-PAGE with less than stoichiometric iron for a protein possessing two [4Fe-4S] clusters (Figure 5.1).(1, 8, 9) Figure 5.1. UV-vis spectroscopy of pre and post reconstituted HydE (83 µM). The arrow denotes the increase in the 420 nm due to the loading of FeS clusters upon reconstitution. Left embedded image is SDS-PAGE gel representing the relative purity of HydE following purification. Right embedded image shows the intense brown color of reconstituted HydE. The protein was reconstituted with exogenous iron and sulfide under reducing conditions yielding protein with 8.1 ± 0.5 Fe/protein over three separate purifications and reconstitutions of HydE. UV-vis absorption spectroscopy of HydE and S- adenosylmethionine (SAM) employed a Cary 60 spectrophotometer in a 1.4 mL 96 anaerobic cuvette. The dark brown reconstituted protein shared a marked increase in absorbance at 420 nm (Figure 5.1) indicative of increased occupancy of FeS clusters in the purified protein after removal of adventitiously bound iron.(10-12) Preparation of HydE Variants For the generation of the C. acetobutylicum HydE variants the Agilent Technologies QuikChange Lighting Multi Site-Directed Mutagenesis Kit was employed. The manufacture’s recommendations were used with 0.5 µL of the QuikSolution per 25 µL of reaction and the previously described pET23b-C.a.HydE template (Chapter 3). The mutagenic primers used with the specific base substitutions bolded and underlines were GAGTACAGAAAATCTGGAGCTGATAGATATTTAATAGCAATAGAAACAACA GATAAAG and CAGAAAATCTGGAGCTGATAGATATTTAATAAAAATAGAAACAACAGATAA AGAACTG for the R159A and R159K variants, respectively. The mutagenesis protocol produces single stranded plasmids that were transformed into Agilent XL-10 Gold Ultracompetent cells. The sequence verified constructs were transformed into the overexpression E. coli strain BL21(DE3)-RIL. The overexpression, purification, and reconstitution steps were performed anaerobically in a manner similar to the wild-type C. acetobutylicum HydE previously described in Chapters 2 and 4. The purified variants possessed 9.8 ± 0.5 and 9.1 ± 0.4 Fe/protein for R159A and R159K, respectively. 97 Electron Paramagnetic Resonance Spectroscopy All EPR samples were prepared in a strictly anaerobic environment maintained by a MBRAUN chamber (O2 ≤ 1 ppm). Solutions were prepared using buffer and water degassed on a Schlenk line by three cycles of 10 minutes of vacuum followed by backfill with nitrogen. The water mass lost from the buffers by evaporation was replaced with degassed water. Given the potential for interference with the high concentration of potassium ions (500 mM) with the putative cation binding pocket, the C. acetobutylicum HydE protein was buffer exchanged into 25 mM Tris-HCl pH 8.0 removing most KCl and glycerol. The protein was stable in this buffer after dialysis but rapidly precipitated after a single freeze/thaw. A saturated thallium (I) chloride solution stock was prepared to approximately 10 mM. The 250 µL (final volume) EPR samples were prepared with 100 µM HydE, 1 mM S-adenosylmethionine (SAM), ~5 mM TlCl, and 3 mM sodium dithionite. X-band, CW spectra were obtained using a Bruker ER-200D-SRC spectrometer with a liquid helium cryostat and Oxford Instruments temperature controller. EPR spectra were collected with the following parameters unless noted otherwise: 1 mW power, 20.48 ms time constant/conversion time, 1 x 104 receiver gain, 100 kHz modulation frequency, 12-16 K temperature, and a modulation amplitude of 10 G. OriginPro (Version 8.5) was used to baseline correct spectra and EasySpin 4.5.0 was used to assign g-values.(13) 98 HydE Turnover Assays C. acetobutylicum wild type and R159A/K variant reconstituted proteins were assayed for SAM cleavage and 5’-deoxyadenosine (dAdo) production rates in the presence of 25 µM HydE, 0.5 mM SAM, 2.5 mM thiol, and 2 mM sodium dithionite in the 25 mM HEPES pH 8.0, 500 mM KCl, and 5% glycerol (w/v) buffer. The assays were incubated at 37 ˚C for 30 minutes following the addition of sodium dithionite and were quenched by addition of 10% (v/v) 400 mM acetic acid buffer pH 4.5. The samples were placed on ice for about 1 hr followed by centrifugation for 10 min at 4 ˚C to remove the precipitated protein. The quantities of dAdo produced were measured by reverse phase chromatography on an Agilent Technologies 1100 series HPLC using a Phenomenex Kinetix 5 u PFP 100A column by a method that has previously been described.(1) The mobile phases were (A) water and (B) acetonitrile with 0.1% acetic acid. In short, 10 µL of the reaction supernatant was loaded on a 2% B equilibrated column and washed with 2% B for 3.3 min. A linear gradient run up to 60% B over 11 min caused dAdo to elute at 6.8 min. The dAdo (λmax = 254 nm) concentration was determined using a standard curve. Production and Detection of Glyoxylate Aliquots from dAdo production/SAM cleavage assays explained above were subjected to a derivatization method in order to detect any glyoxylate as previously described with minor modifications.(14, 15) In addition to mercaptopyruvate and the full assay complement, controls without substrate, with cysteine, and without HydE were 99 tested. The supernatant (10 µL) from 30 min quenched reaction aliquots were diluted 5- fold with 40 µL of assay buffer, acidified by addition of 100 µL of 500 mM HCl, and derivatized with 50 µL of 10 mg/mL o-phenylenediamine. The solution was incubated at 95 ˚C for 10 min prior to addition of 120 µL 1.25 M NaOH. A standard curve with 0 to 25 µM glyoxylate was prepared for quantification using glyoxylic acid monohydrate from Sigma-Aldrich. The samples were flash frozen with liquid nitrogen and thawed just prior to HPLC-FLD analysis. A Luna 5u 100A Phenomenex C18 column was used on an Agilent Technologies 1100 series HPLC linked to an Agilent Infinity 1260 fluorescence detector for separation and quantitation of the derivatized glyoxylate product 2-quinoxalinol. The HPLC solvents were (A) 100 mM NH4HCO3 in water and (B) acetonitrile. The 35 min HPLC- FLD run included a 5 min wash with 5% B followed by a linear gradient to 60% B over 15 minutes. The column was then washed with 100% B for 5 min and the column was equilibrated with 5% B for 10 min prior to the following injection. The fluorescence detector monitored a 420 nm emission during excitation at 340 nm. A peak corresponding to the derivatized glyoxylate standard 2-quinoxalinol eluted mid-gradient at 15 minutes. Assays investigating the lyase activity of HydE on L-cysteine and L- homocysteine required modifications of the standard turnover assays described in the previous section. Given the interference of KCl and glycerol with the LC-MS signal, HydE was buffer exchanged into a 25 mM NH4HCO3 pH 8.0 buffer. Testing the hypothesis that HydE may convert L-homocysteine and L-cysteine to L-alanine and 100 glycine, respectively a normal phase chromatography separation prior to analysis by mass spectrometry was employed. Given the lower quantitation threshold for these amino acids than derivatized glyoxylate with HPLC-FLD the assay components’ concentrations were increased to 100 µM HydE, 300 µM SAM, 10 mM substrates, and 3 mM NaDT. The reactions were allowed to react for 2 hrs at 37 ˚C prior to being quenched with an equal volume of acetone. The precipitated protein was removed by centrifugation at 14K rpm at 4 ˚C prior to LC-MS analysis. A volume of 2 µL of the supernatant was injected onto a Cogent Diamond Hydride 100A 4 µm 150 x 2.1 mm normal phase column equilibrated in 80% solvent B (acetonitrile) and 20% solvent A (water) both containing 0.1% trifluoroacetic acid and washed for 1 min at this ratio. A linear gradient from 80% down to 50% B of 5 min was sufficient to elute both glycine and alanine at around 1.8 min into the gradient. The column was washed at 50% B for 2.5 min and reequilibrated into 80% B for 1.5 min prior to injection of the following sample. Turnover assays were designed given the detection limit of 20 µM alanine and glycine with a 2 µL injection. Mapping Conserved Residues and Internal Volume of HydE Several topographic and bioinformatics investigations were made using the Thermotoga maritima HydE crystal structure solution PDB ID: 3IIZ.(8) To map the void space within HydE, 3IIZ was uploaded to the CASTp server(16) generating a .pyc file that could be opened in the software suite UCSF Chimera.(17) A 1.4 Å space filling sphere size was chosen to model the solvent exposed and interior cavities of HydE. 101 To measure the relative conservation of individual HydE residues and map them to the crystal structure a Basic Local Alignment Search Tool (BLAST) search was conducted to identify and retrieve the top 500 sequences similar to T. maritima HydE. The BLAST result was associated with the T. maritima HydE crystal structure solution in UCSF Chimera allowing the selection of residues by their percent conservation. Results HydE R159 Variants Slow SAM Cleavage The substrate binding pocket lies at the top of a large internal cavity inside the complete (βα)8 TIM barrel fold of HydE.(8) The active site houses SAM at the top of the barrel and a channel extends to the bottom of the barrel. Several conserved residues that are speculated to be involved in substrate binding and product delivery line the interior of the barrel. The residue R159 is strictly conserved and likely binds an anionic moiety of HydE’s substrate given that two chloride ions were crystallized in the protein in a manner similar to a carboxylic acid (Figure 5.2). The HydE variants R159A and R159K exhibited a much slower rate of dAdo production (20% and 40% of WT activity, respectively) in the absence of potential cosubstrates (Figure 5.2). The relative trend in stimulation of SAM cleavage by cysteine (Cys), mercaptopyruvate (MP), and coenzyme M (CoM) was similar to that observed in the WT enzyme, although in all cases the variants showed reduced rates.(1) These results indicate that R159 plays an important role in coordination of the substrate for HydE but other positively charged moieties my contribute to the anionic binding pocket. 102 Figure 5.2. 5’-Deoxyadenosine produced by WT HydE (7.6 Fe/protein) and the R159A (9.8 Fe/protein) and R159K (9.1 Fe/protein) variants (25 µM). SAM (0.5 mM) was present in all assays, and for each protein a negative control and putative substrates (2.5 mM) were tested. The inserted figure from PDB ID: 3IIZ shows the relative location of R159A, the chloride ions, SAM, and the [4Fe-4S] cluster in the active site of Thermotoga maritima HydE. Labeling is as follows: carbon (green), nitrogen (blue), oxygen (red), sulfur (yellow), iron (orange), and chlorine spheres (green). HydE Mercaptopyruvate Lyase Activity Mercaptopyruvate, L-cysteine, and L-homocysteine were all screened as substrates for HydE and investigations were made to find the potential products of catalysis. The hypothesis that HydE could catalyze H-atom abstraction from mercaptopyruvate, L-cysteine, and L-homocysteine to produce thioformaldehyde and glyoxylate, glycine, and L-alanine, respectively, was tested by looking for these fragments following turnover (Figure 5.3). Neither glycine nor L-alanine was observed by mass spectrometry in the assays of L-cysteine and L-homocysteine. The detection of theses small polar molecules, however, is difficult and their empirically determined detection limits of ~20 µM may have not allowed for observation. In contrast, the assay 103 of mercaptopyruvate turnover by HydE revealed levels of glyoxylate that were significantly increased over background (Figure 5.4). Approximately two molecules of glyoxylate per HydE after 30 min were produced in the presence of mercaptopyruvate upon incubation. These results support the hypothesis that a small molecular weight thiol, in particular mercaptopyruvate, serves as the substrate for HydE. Figure 5.3. Boxed Area: the proposed mechanism for H-atom abstraction by HydE to produce thioformaldehyde (S=CH2), 5’-deoxyadenosine (dAdoH), and a fragment. The potential substrates tested, and their predicted products, are shown below the box. Only the product from mercaptopyruvate lyase activity was observed. 104 Figure 5.4. HydE production of glyoxylate. The production of glyoxylate after 30 min of addition of mercaptopyruvate (MP)/L-Cys (2.5 mM) to HydE with 7.6 Fe/protein (25 µM) with SAM (500 µM) and sodium dithionite (2 mM) under turnover conditions. HydE only, HydE + L-Cys (n = 1), and HydE + mercaptopyruvate (MP) all served as negative controls. Thallium Nuclear Spin Interaction with FeS Cluster Signal Both stable isotopes 203Tl and 205Tl possess I = ½ nuclear spin; when these bind close to a paramagnetic center, splitting of the EPR signals into doublets can be observed.(18) The superhyperfine interaction of thallium with paramagnetic clusters makes it a powerful tool to probe the distances of monovalent ion binding sites to EPR active paramagnetic centers.(18, 19) Upon addition of thallium salts to HydE with or without SAM, the EPR signal was dramatically modified (Figure 5.5). The broadening observed in the features is most likely due to peak splitting caused by interaction of the 105 nuclear spin of the thallium with the unpaired electrons of the [4Fe-4S]+ cluster. The most intense signal corresponding to the N-terminal cluster with SAM bound was the easiest feature to model. The splitting of +/- 23 gauss from the parent 3500 Gauss signal decreased the parent signal by 50%, with the overall spectrum appearing to be the result of an overlap of two [4Fe-4S]+ signals, only one of which is close to Tl+ (Figure 5.5B). These observations are indicative of a monovalent cation binding site near to the N- terminal radical SAM cluster. We postulated that this cation binding site could serve as a site for binding ammonium for use in synthesizing the DTMA bridge by reaction with thioformaldehyde. In order to probe the possibility of ammonium binding to the cation binding site, we carried out a competition experiment to determine whether ammonium can displace thallium(I) from this site. Upon addition of ammonium to 100 µM HydE buffer exchanged into 100 mM Tris-NO3- pH 8.0, 1 mM SAM, 1 mM TlCl, and 3 mM sodium dithionite the EPR signal (g = 2.01, 1.88, 1.83) sharpened indicating that thallium was being displaced (Figure 5.5). Although relatively high concentrations of ammonium were required to achieve this displacement, thallium’s persistence and resistance to substitution of potassium has previously been noted for other metalloenzymes.(18, 19) Further spectroscopic investigations including ENDOR spectroscopy could give us a more accurate distance of the Tl+ to the paramagnetic [4Fe-4S]+ cluster. 106 Figure 5.5. C. acetobutylicum HydE with addition of Tl+. A. EPR spectra of reduced HydE with 7.6 Fe/protein (black trace) (100 µM) and reduced HydE with addition of Tl+ (red trace) (5 mM). B. Same as in panel A but with addition of exogenous SAM (1 mM). C. Titration with 0.5 mM (blue trace), 5.0 mM (yellow trace), and 20 mM (red trace) ammonium into reduced HydE (100 µM), SAM (1 mM), and Tl+ (1 mM). The EPR parameters for A-C are described in the methods section. D. Distance of 5’ carbon of SAM to putative cation binding site in HydE (PDB ID: 3IIZ). Discussion Putative Active Site Residues While PylB(20) and NosL,(21) two radical SAM enzymes similar to HydE (Chapters 1 and 4), have published crystal structures with substrate/product bound, HydE has never 107 been crystallized with a substrate molecule as its identity is unknown. Our recent publication (Chapter 4) reported a multidisciplinary approach that implicated low molecular mass thiols as candidates for HydE’s substrate.(1) In silico docking studies indicate that these thiols have an anionic moiety that can salt bridge with HydE Arg159 (T. maritima and C. acetobutylicum numbering). In addition, the crystal structure was found to contain two chloride ions occupying roughly the space of a carboxyl group of an undefined substrate.(8) In the work described in this chapter, this arginine was mutated conservatively to a lysine and non-conservatively to an alanine. While neither of these residues likely affect the binding of SAM, a mutation that effects substrate binding of radical SAM enzymes would likely lower SAM cleavage rates. We hypothesized that the R159K variant protein would have much lower SAM cleavage/5’-deoxyadenosine production rates and the R159A variant would have little or no activity.(19) Our results show that on average across the board R159K had 40% of WT SAM cleavage activity, while the R159A exhibited approximately 20% of WT activity. While the SAM cleavage rates were affected less than we might have expected, further investigation into the active site revealed that there are other positive moieties that likely contribute to the total anionic binding pocket; these include the backbone nitrogens from Thr269 and Ala270 (T. maritima numbering) located <3.5 Å from the cocrystallized chloride ions (Figure 5.6). In addition, the oxygen on the Tyr306 phenol group is only 3.5 Å away, perhaps with the hydrogen aiming towards the anion binding pocket acting as a H-bond donor to the negatively charged moiety of the substrate. These additional positive moieties may be sufficient to stabilize the thiols in the active site albeit at a compromised affinity. The 108 decrease in the SAM cleavage rates in the absence of thiols may be the result of changes in the electronic environment of the [4Fe-4S] cluster and perturb either the reduction of the cluster or the transfer of the electron to the positively charged sulfonium of SAM. Figure 5.6. Active site anion binding pocket of T. maritima HydE (PDB ID: 3IIZ) with residues highlighted that likely contribute to the positively charged pocket. Labeling is as follows: carbon (green), nitrogen (blue), oxygen (red), sulfur (yellow), iron (orange), and chlorine spheres (purple). Mechanism for Lyase Activity We recently proposed that the synthesis of the dithiomethylamine (DTMA) bridge of the H-cluster might form via the result of HydE generating highly reactive thioformaldehyde units from a low molecular mass mercaptan, such as cysteine or mercaptopyruvate.(1) The observation that glyoxylate is produced enzymatically by 109 HydE in the presence of mercaptopyruvate supports the mechanism shown in Figure 5.4. Detection of the thioformaldehyde product itself is challenging due to its high reactivity.(22) Two successive HydE turnover events may produce two thioformaldehyde molecules that would condense with ammonia/ammonium in the interior of the TIM barrel to produce the dithiomethylamine bridge, in a manner reminiscent of the in vitro synthesis of H-cluster analogs (Figure 5.7).(23) Figure 5.7. Upper Box: In vitro synthesis of H-cluster analogs by condensation of formaldehyde and ammonia on a 2Fe scaffold.(23) Lower Box: Proposed in vivo synthesis of DTMA bridge from thioformaldehyde condensation with ammonia. DTMA could subsequently form the H-cluster by joining two iron-diatomic species proposed by Kuchenreuther et al.(24) (bottom) or by alkylation of an existing [2Fe-2S] cluster presumably bound to HydF(25) (top). The observation that glyoxylate is enzymatically produced under HydE turnover conditions when mercaptopyruvate is present supports the hypothesis that a low molecular mass thiol is acted upon by HydE. It also favors the two hypotheses that lyase 110 activity on the substrate produces thioformaldehyde and the in vivo mechanism for synthesis of the DTMA bridge occurs in a manner similar as shown in Figure 5.7. EPR investigations with HydE in the presence of Tl1+ provide additional support for this mechanism by pointing to an ammonium binding pocket in the active site. The EPR spectra show strong evidence of interaction of the nuclear spin of thallium with the paramagnetic [4Fe-4S]+ cluster both in the presence and absence of SAM (Figure 5.3A and B). Further, the effects of the thallium nuclear coupling are diminished in the presence of ammonium ion (Figure 5.5C), indicating that ammonium can displace thallium from this binding site. The proposed site for binding the thallium or ammonium is the negatively charged pocket near (~ 8 Å) the active site of HydE (Figure 5.5D), which could support the ammonium ion used for synthesis of the DTMA bridge. In the T. maritima HydE crystal structure PDB ID: 3IIZ, this negatively charged pocket is roughly 10 Å from the SAM binding N-terminal cluster which is likely too far to observe thallium coupling by EPR, but could still be close enough to bind a substrate ammonium.(18) The affinity for ammonium appears to be less than that of thallium given the relative concentrations of these cations in the titration experiments (Figure 5.5C); however, intracellular concentrations of ammonium in some bacteria have been found to be as high as 138 mM, which may explain the apparent low affinity for ammonium.(26) Also, thallium has been reported to have a greater affinity for cation binding sites than the appropriate biologically relevant cation as it is difficult to displace.(18) 111 Key C-terminal Active Site Residues Conservation analysis of the amino acid sequence of homologous proteins often reveals residues important to ligand binding, protein-protein interactions, structural stability, and active site catalysis.(27, 28) Alignment of the top 500 most similar sequences to T. maritima HydE can be used to highlight the most conserved residues. This method is especially powerful when the conservation filters are applied to a crystal structure solution and residues of relative conservation are highlighted. Given the possibility for sequencing errors or misalignments of the amino acid sequences, we defined residues with 99.3% or greater conservation as highly conserved. The tertiary structure for all known radical SAM enzymes is that of a complete or partial TIM barrel.(29, 30) The TIM barrel is composed of alternating βα units that fold to create an environment that largely excludes the bulk solvent shielding the active site radical from side reactions. The purple “cast” of the interior space (Figure 5.8B) in HydE is composed of 1.4 Å spheres occupying a volume of 1744.8 Å3. A large portion of the upper chamber of this interior space is occupied by the [4Fe-4S] cluster and SAM cofactors as represented in Figure 5.8A. The most highly conserved residues (≥99.3%) of HydE are depicted as sticks in Figure 5.8A, C, and D. A majority of these highly conserved residues are located in the central portion of the structure lining the interior of the TIM barrel. The amino acids that bind the SAM and the [4Fe-4S] cluster cofactors are highly conserved, chiefly being the CX3CX2C motif and Asp161 (T. maritima HydE numbering). 112 Figure 5.8. HydE Crystal Structures from PDB ID: 3IIZ and 3CIX. A. Side view of cartoon structures with 99.3% and higher conserved residues, thiocyanate (SCN-), SAM, and [4Fe-4S] cluster are explicitly shown. B. Side view of cartoon structure with interior volume represented as a purple cast made by 1.4 Å spheres. C. Bottom view of Figure 5.8A. D. Close up view of highly conserved residues surrounding thiocyanate. An alternate view of this site is given in Figure 5.9. The color-coding is tan and gray for carbon, blue for nitrogen, red for oxygen, yellow for sulfur, and brown for iron. The “bottom” of the interior of the TIM barrel of HydE was found to bind thiocyanate with high affinity in a small molecule soak of HydE crystals.(8) While the relevance of this thiocyanate binding is yet unknown, it is quite interesting that the residues that surround thiocyanate are highly conserved (Figures 5.8A, C, and D). Figure 5.8D and Figure 5.9 are representations of the highly conserved residues at the bottom of the HydE TIM barrel. Six of the highly conserved residues can be divided into two 113 nearly symmetrical triads with a methionine, arginine, and threonine. The arginines of the triads are solvent accessible and may be involved in a gating mechanism for the delivery of the DTMA bridge, presumably to HydF.(31) Also, the symmetry of the residues (Figure 5.9) at the lower access point of HydE may serve to stabilize the symmetrical product or an intermediate in DTMA synthesis (Figure 5.7). Figure 5.9. T. maritima HydE crystal structure PDB ID: 3CIX side (A) and bottom zoomed in (B) views. The tertiary structure is in semitransparent green. SAM, thiocyanate, and the [4Fe-4S] cluster are colored light blue, magenta, and orange, respectively. Six residues that can be divided into two semisymmetrical conserved triads at the bottom of the TIM barrel. The triads consist of Met224/Arg155/Thr134 and Met291/Arg54/Thr103. The residue Thr103 has a low occurrence of the conservative mutation to a serine. The sulfurs have multiple conformations but the spheres shown encompass both. Yellow spheres represent the sulfurs in the methionines. Blue spheres represent the central carbons of the guanidinium groups of the arginine residues. Red spheres represent the oxygens of the alcohol groups of the threonine residues. Conclusions Building on the multidisciplinary study that proposed the substrate for HydE is a low molecular mass thiol,(1) several further key observations have been made that add to this hypothesis and provide additional insight into the second active site of HydE where thioformaldehyde condenses with ammonia. Using sequence alignment to identify the 114 residues with the highest level of conservation, it was revealed that two triads of Arg/Met/Thr might be the site of the deliverable DTMA bridge to HydF. The symmetry of these two residue triads hints at a HydE product staging area of the presumably symmetrical DTMA precursor. Through mutagenesis of the anionic binding pocket near the site of radical catalysis we showed a marked decrease in SAM cleavage supporting the hypothesis that this pocket is important to substrate binding. While several thiols have been shown to stimulate production of 5’-deoxyadenosine from SAM cleavage and mediate incorporation of deuterium from the solvent exchangeable thiols to the 5’ carbon of 5’-deoxyadenosine,(1) we observed a marked stimulation of production of a potential product (glyoxylate) only in the presence of mercaptopyruvate (Figure 5.4). The mercaptopyruvate lyase activity we observed by HydE is the first report of product formation by HydE. All theses findings support the hypotheses that (i.) HydE’s substrate is mercaptopyruvate, (ii.) the DTMA bridge precursor is formed by the condensation of ammonia and two lyase fragments, and (iii.) the lower active site area serves as the location of DTMA precursor synthesis and the antechamber for delivery to HydF. Further investigations with the other maturation partners HydG and HydF will refine the details of transfer of the DTMA bridge during the construction of the H-cluster. 115 References 1. Betz, J. N., Boswell, N. W., Fugate, C. J., Holliday, G. L., Akiva, E., Scott, A. G., Babbitt, P. C., Peters, J. W., Shepard, E. M., and Broderick, J. B. (2015) [FeFe]- Hydrogenase Maturation: Insights into the Role HydE Plays in Dithiomethylamine Biosynthesis, Biochemistry 54, 1807-1818. 2. Stephenson, M., and Stickland, L. H. (1931) Hydrogenase: A bacterial enzyme activating molecular hydrogen. I. The properties of the enzyme., Biochem J 25, 205-214. 3. Adams, M. W. W., Mortenson, L. E., and Chen, J. S. (1980) Hydrogenase, Biochimica et biophysica acta 594, 105-176. 4. Peters, J. W., Lanzilotta, W. N., Lemon, B. J., and Seefeldt, L. C. (1998) X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution, Science 282, 1853-1858. 5. Nicolet, Y., de Lacey, A. L., Vernede, X., Fernandez, V. M., Hatchikian, E. C., and Fontecilla-Camps, J. C. (2001) Crystallographic and FTIR spectroscopic evidence of changes in Fe coordination upon reduction of the active site of the Fe- only hydrogenase from Desulfovibrio desulfuricans, J.Am.Chem.Soc. 123, 1596- 1601. 6. Erdem, O. F., Schwartz, L., Stein, M., Silakov, A., Kaur-Ghumaan, S., Huang, P., Ott, S., Reijerse, E. J., and Lubitz, W. (2011) A model of the [FeFe] hydrogenase active site with a biologically relevant azadithiolate bridge: a spectroscopic and theoretical investigation, Angewandte Chemie 50, 1439-1443. 7. Ryde, U., Greco, C., and De Gioia, L. (2010) Quantum refinement of [FeFe] hydrogenase indicates a dithiomethylamine ligand, Journal of the American Chemical Society 132, 4512-4513. 8. Nicolet, Y., Rubach, J. K., Posewitz, M. C., Amara, P., Mathevon, C., Atta, M., Fontecave, M., and Fontecilla-Camps, J. C. (2008) X-ray structure of the [FeFe]- hydrogenase maturase HydE from Thermotoga maritima, J.Biol.Chem. 283, 18861-18872. 9. Rubach, J. K., Brazzolotto, X., Gaillard, J., and Fontecave, M. (2005) Biochemical characterization of the HydE and HydG iron-only hydrogenase maturation enzymes from Thermatoga maritima, FEBS Lett. 579, 5055-5060. 10. Shen, G., Balasubramanian, R., Wang, T., Wu, Y., Hoffart, L. M., Krebs, C., Bryant, D. A., and Golbeck, J. H. (2007) SufR coordinates two [4Fe-4S]2+, 1+ clusters and functions as a transcriptional repressor of the sufBCDS operon and an 116 autoregulator of sufR in cyanobacteria, The Journal of biological chemistry 282, 31909-31919. 11. Lippard, S. J., and Berg, J. M. (1994) Principles of bioinorganic chemistry, University Science Books, Mill Valley, Calif. 12. Ruzicka, F. J., and Frey, P. A. (2007) Glutamate 2,3-aminomutase: a new member of the radical SAM superfamily of enzymes, Biochimica et biophysica acta 1774, 286-296. 13. Stoll, S., and Schweiger, A. (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR, J Magn Reson 178, 42-55. 14. Kriek, M., Martins, F., Challand, M. R., Croft, A., and Roach, P. L. (2007) Thiamine biosynthesis in Escherichia coli: identification of the intermediate and by-product derived from tyrosine, Angew.Chem.Int.Ed Engl. 46, 9223-9226. 15. Driesener, R. C., Duffus, B. R., Shepard, E. M., Bruzas, I. R., Duschene, K. S., Coleman, N. J. R., Marrison, A. P. G., Salvadori, E., Kay, C. W. M., Peters, J. W., Broderick, J. B., and Roach, P. L. (2013) Biochemical and Kinetic Characterization of Radical S-Adenosyl-L-methionine Enzyme HydG, Biochemistry 52, 8696-8707. 16. Dundas, J., Ouyang, Z., Tseng, J., Binkowski, A., Turpaz, Y., and Liang, J. (2006) CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues, Nucleic Acids Res 34, W116-W118. 17. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004) UCSF chimera - A visualization system for exploratory research and analysis, J Comput Chem 25, 1605-1612. 18. Markham, G. D., and Leyh, T. S. (1987) Superhyperfine Coupling between Metal-Ions at the Active-Site of S-Adenosylmethionine Synthetase, Journal of the American Chemical Society 109, 599-600. 19. Kayne, F. J. (1971) Thallium (I) Activation of Pyruvate Kinase, Archives of biochemistry and biophysics 143, 232-&. 20. Quitterer, F., List, A., Eisenreich, W., Bacher, A., and Groll, M. (2012) Crystal structure of methylornithine synthase (PylB): insights into the pyrrolysine biosynthesis, Angewandte Chemie 51, 1339-1342. 21. Nicolet, Y., Zeppieri, L., Amara, P., and Fontecilla-Camps, J. C. (2014) Crystal Structure of Tryptophan Lyase (NosL): Evidence for Radical Formation at the Amino Group of Tryptophan, Angewandte Chemie. 117 22. Johnson, D. R., Powell, F. X., and Kirchhof.Wh. (1971) Microwave Spectrum, Ground State Structure, and Dipole Moment of Thioformaldehyde, J Mol Spectrosc 39, 136-&. 23. Li, H. X., and Rauchfuss, T. B. (2002) Iron carbonyl sulfides, formaldehyde, and amines condense to give the proposed azadithiolate cofactor of the Fe-only hydrogenases, Journal of the American Chemical Society 124, 726-727. 24. Kuchenreuther, J. M., Myers, W. K., Suess, D. L. M., Stich, T. A., Pelmenschikov, V., Shiigi, S. A., Cramer, S. P., Swartz, J. R., Britt, R. D., and George, S. J. (2014) The HydG Enzyme Generates an Fe(CO)(2)(CN) Synthon in Assembly of the FeFe Hydrogenase H-Cluster, Science 343, 424-427. 25. Shepard, E. M., McGlynn, S. E., Bueling, A. L., Grady-Smith, C. S., George, S. J., Winslow, M. A., Cramer, S. P., Peters, J. W., and Broderick, J. B. (2010) Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold, Proceedings of the National Academy of Sciences of the United States of America 107, 10448-10453. 26. Gerth, K., and Reichenbach, H. (1986) Determination of Bacterial Ammonia Pools Using Myxococcus-Virescens as an Example, Anal Biochem 152, 78-82. 27. Capra, J. A., and Singh, M. (2007) Predicting functionally important residues from sequence conservation, Bioinformatics 23, 1875-1882. 28. Lopez, G., Valencia, A., and Tress, M. L. (2007) firestar - prediction of functionally important residues using structural templates and alignment reliability, Nucleic Acids Res 35, W573-W577. 29. Shisler, K. A., and Broderick, J. B. (2012) Emerging themes in radical SAM chemistry, Curr Opin Struc Biol 22, 701-710. 30. Vey, J. L., and Drennan, C. L. (2011) Structural insights into radical generation by the radical SAM superfamily, Chemical reviews 111, 2487-2506. 31. Shepard, E. M., Mus, F., Betz, J. N., Byer, A. S., Duffus, B. R., Peters, J. W., and Broderick, J. B. (2014) [FeFe]-Hydrogenase Maturation, Biochemistry. 118 CHAPTER 6 [FeFe]-HYDROGENASE MATURATION Contribution of Authors and Co-Authors Manuscripts in Chapters 1, 6, 7 Author: Eric M. Shepard Contributions: Researched the literature, compiled co-authors’ contributions, and prepared the manuscript and figures. Co-Author: Florence Mus Contributions: Compiled literature and prepared parts of an early draft of the manuscript. Co-Author: Jeremiah N. Betz Contributions: Researched the literature and prepared the manuscript and figures. Co-Author: Amanda S. Byer Contributions: Researched the literature and prepared the manuscript and figures. Co-Author: Benjamin R. Duffus Contributions: Researched the literature and prepared the manuscript and figures. Co-Author: John W. Peters Contributions: Researched the literature and aided in preparation of the manuscript and figures. Co-Author: Joan B. Broderick Contributions: Researched the literature and aided in preparation of the manuscript and figures. 119 Manuscript Information Page Authors: Eric M. Shepard, Florence Mus, Jeremiah N. Betz, Amanda S. Byer, Benjamin R. Duffus, John W. Peters, and Joan B. Broderick Journal: Biochemistry Status of Manuscript: ____ Prepared for submission to a peer-reviewed journal ____ Officially submitted to a peer-review journal ____ Accepted by a peer-reviewed journal _X__ Published in a peer-reviewed journal Publisher: American Chemical Society Issue in which manuscript appears: Volume 53, 4090-4104 (2014) 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 CHAPTER 7 INVESTIGATING THE ACTIVE SITES OF HydG Introduction In a study of insertional Chlamydomonas reinhardtii mutants, three gene products, HydE, HydF, and HydG, were found necessary to mature the structural protein HydA also known as [FeFe]-hydrogenase.(1) Examination of HydE and HydG amino acid sequences found they both contain CX3CX2C motifs indicative of radical SAM enzymes.(1) Further biochemical and spectroscopic studies confirmed that they were radical SAM enzymes and revealed much more about HydG’s role in the maturation of HydA.(2) HydG contains two FeS clusters intimately involved in the breakdown of tyrosine into the CO and CN- ligands that decorate the active site H-cluster of HydA.(3-5) The reaction catalyzed by the HydG is thought to be initiated by electron transfer from the reduced N-terminal [4Fe-4S] cluster to S-adenosylmethionine to produce the highly reactive deoxyadenosyl radical intermediate. The work described in this chapter was designed to probe subsequent steps in catalysis. Our results support a mechanism in which this deoxyadenosyl radical abstracts an H-atom from the amine group of tyrosine bound near the N-terminal cluster, with this substrate radical undergoing homolytic cleavage to generate dehydroglycine ad a p-hydroxybenzylic radical. Data is also presented that point to key residues in the HydG’s active site that are required for substrate binding and catalysis. Finally, EPR spectra are presented that are interpreted as 136 the C-terminal cluster being a site differentiated [4Fe-4S] cluster that is likely involved in the subsequent production of CO and/or CN-. Materials and Methods Mutagenesis of Active Site Residues in C. acetobutylicum HydG Several Clostridium acetobutylicum hydG mutant constructs were generated using previously prepared pCDFDuet-hydG constructs by two different mutagenesis methods.(6) For the generation of the C. acetobutylicum HydG S338V and S338D variants the Agilent Technologies QuikChange Lightning Multi Site-Directed Mutagenesis Kit was employed. The manufacturer’s recommendations were employed with 0.5 µL of the QuikSolution per 25 µL of reaction and the previously mentioned pCDFDuet-hydG template. The mutagenic primers used for the S338V and S338D were: GTGCTAGAACCACCAACTATTTGAGATATACCGAGTTCAAGTACGC and GTGCTAGAACCACCATCTATTTGAGATATACCGAGTTCAAGTACGC, respectively. This technique produced single stranded plasmids that were transformed into Agilent XL-10 Gold Ultracompetent cells. The sequence verified constructs were then transformed into the overexpression E. coli strain BL21(DE3)-RIL. The constructs for the HydG variants S338A, Q360E, and Y91F were created from the same WT construct, but by using the Gibson cloning mutagenesis technique.(7) We employed this technique to join three DNA fragments with complementary ends using a three enzyme cocktail to produce a finished construct coding for the desired variant protein. The WT construct was linearized and the HydG gene was removed by 137 double digestion with the New England Biolabs restriction enzymes BamHI and HindIII in NEBuffer #2 according to the manufacturer’s instructions. The enzymes were then permanently heat-inactivated by incubation at 80 ˚C for 20 min. The linearized pCDFDuet (Fragment 1) was subsequently used for all three variants prepared employing the Gibson technique. The second and third fragments for each mutant were prepared using the PCR conditions listed in Figure S2 and the two sets of relevant primers listed in Figure S1. The PCR fragments and linearized template were assembled in the triple enzyme and reagent cocktail that included a polymerase, ligase, and 5’-exonuclease (Figure S2) by incubating the reaction at 50 ˚C for 60 min. The assembled construct was immediately transformed into the E. coli strain Nova Blue chemically competent cells according to the manufacturer’s directions. Constructs were sequence verified for the correct insert prior to a new transformation and overexpression, the protocols for which are as described in Chapter 2. The two additional C. acetobutylicum HydG variants were obtained by utilizing previously constructed plasmids. The first was the HydG∆CTD variant in which the C- terminal domain is truncated so that only the radical SAM [4Fe-4S] cluster is present.(4) The second HydG variant, termed HydG-NTM, had C96A, C100A, and C103A mutations which effectively removed the radical SAM binding domain.(4) Growth, Purification, and Reconstitution of HydG Protein for biochemical and spectroscopic characterization was produced for each variant as described in Chapter 2. The only deviation from the protocol was the 138 expression of the HydG variants HydG-∆CTD and HydG-NTM in which only half of the exogenous Fe3+ and S2- were added during reconstitution (only a 3-fold excess over protein) given the variant proteins could bind only a single [4Fe-4S] cluster. All subsequent protein purification, reconstitution, and assays were carried out using a buffer that contained 500 mM KCl, 25 µM HEPES pH 8.0, and 5% glycerol unless otherwise described. EPR of Rapidly Quenched HydG Turnover Samples To capture a radical intermediate that HydG transiently produces during enzymatic turnover, samples were prepared by rapidly mixing two reaction components and flash freezing the reaction for analysis by EPR. Volumes of 125 µL of component A (200 µM HydG, 6 mM L-tyrosine, 6 mM S-adenosylmethionine, and buffer) and component B (6 mM sodium dithionite and buffer) were injected through separate syringes into quartz EPR tubes. Both WT HydG (6.5 Fe/protein) and HydG∆CTD (2.4 Fe/protein) samples were made. The mixed components were allowed to incubate for 5- 30 seconds after which they were flash frozen in liquid propane. Collection of EPR spectra, baseline correction, and g-value assignments were conducted as previously described in Chapters 4 and 5. EPR of Cyanide Treated HydG-NTM HydG-NTM, the variant without the three cysteine motif that binds the radical SAM [4Fe-4S] cluster, was treated with cyanide in a manner similar to that published by Roach and coworkers.(8) Two 200 µL EPR samples were made with 150 µM HydG- 139 NTM and 10 mM sodium dithionite in the standard buffer with and without 3 mM KCN. The samples were flash frozen in liquid nitrogen and EPR spectra were collected and analyzed in a manner described in Chapters 4 and 5. HydG Turnover Assays and Product Quantitation Two sets of HydG turnover assays were conducted while probing the substrate specificity and binding pocket of HydG. In the first set, L-tyrosine analogs (1 mM) including N-methyl-L-tyrosine, O-methyl-L-tyrosine, 5-fluoro-L-phenylalanine, 4- amino-L-phenylalanine, phenylalanine, and tryptophan (Figure 7.1) were incubated with C. acetobutylicum HydG (40 µM), SAM (1 mM), and sodium dithionite (5 mM) in a 50 mM HEPES pH 8.0, 300 mM KCl, and 5% glycerol buffer for 30 min at 37 ˚C in an MBraun anaerobic chamber (≤ 1 PPM O2). The assay samples were removed and the reaction was stopped by the addition of one part (v/v) 1 M acetate buffer pH 4.5 to nine parts (v/v) assay solution. The acidified assay mixture was cooled on ice for an hour and then centrifuged for 10 min at 4 ˚C to remove the precipitated protein. The small molecules in the supernatant were analyzed by HPLC as described in Chapter 5, and in addition the production of p-cresol was monitored by absorbance at 280 nm and comparison to a set of p-cresol standards. A second set of turnover assays were conducted with the suite of HydG S338 variants. The HydG S338A/S338V/S338D variants all overexpressed well and when reconstituted contained 6.9, 7.5, and 6.7 Fe/protein, respectively. These iron-loading values were in good agreement with the WT HydG protein (6.5 Fe/protein) that was used 140 as a control. These mutations were selected to i) test the hypothesis proposed in a recent paper that HydG’s initial H-atom abstraction occurs at the amine nitrogen of substrate tyrosine(9) and ii) engineer the active site of HydG to accommodate alternate amino acids as substrates. Figure 7.1. Tyrosine analogs used in HydG turnover assays. To test the variant proteins’ SAM cleavage rates, and therefore their functionality, turnover assays were conducted with 40 µM protein, 1 mM SAM, 1 mM substrate, and 5 mM sodium dithionite in 25 mM HEPES pH 8.0, 500 mM KCl, and 5% glycerol buffer. The substrates that were used in the assays with the WT and each variant protein were L- tyrosine, L-phenylalanine, and L-histidine. After incubation at 37 ˚C for 2 hr, the reaction was quenched and the protein was destabilized by a 10% addition of 1 M acetate 141 buffer of pH 4.5. The precipitated protein was then removed by centrifugation and the supernatants were analyzed by HPLC as described above. Results Capturing a Radical Intermediate of the Tyrosine Lyase HydG By using a hand-mixing and rapid-freezing technique, we were able to generate the p-hydroxybenzylic radical species previously reported for the S. oneidensis HydG(10) in our C. acetobutylicum HydG. Interestingly, the EPR signal we observe for this radical intermediate is of considerably higher intensity than that initially reported by Kuchenreuther et al.(10) By preincubation of HydG with both SAM and L-tyrosine, rather than just L-tyrosine as done by Kuchenreuther et al., a radical signal of greater intensity could be observed as early as 5 sec, which is the limit of our mixing and flash freezing technique. An EPR spectrum of this radical intermediate is shown in Figure 7.2; the radical signal is isotropic and centered at g = 2 while the contributions of the C-terminal cluster and the N-terminal-SAM bound cluster can also be observed.(4) We have also generated this intermediate using the HydG∆CTD variant that lacks the C-terminal domain and its associated iron-sulfur cluster. In Figure 7.2, the lower set of three EPR spectra for HydG∆CTD under turnover conditions indicate that the kinetics of tyrosine lyase activity for the truncated variant protein may be slower than for the WT protein since the intermediate radical signal (g = 2.0) is of much lower intensity over all recorded time periods. The low field region of the EPR spectra for HydG shown in Figure 7.3 under turnover conditions, represented in Figure 7.3, is clearly devoid of signals other 142 than a minor feature at g = 4.3 that is due to adventitiously bound high spin ferric iron commonly referred to as “junk iron.” Britt and coworkers have observed additional spectral features around g = 4.3 that they have in separate papers attributed to both paramagnetic linear [3Fe-4S] or [5Fe-5S] clusters.(8, 10) Figure 7.2. EPR spectra during turnover showing the generation of the p- hydroxybenzylic radical by HydG. Top three spectra: WT HydG (100 µM) turnover of tyrosine (3 mM) with SAM (3 mM) initiated by addition of sodium dithionite (3mM) and flash frozen in liquid propane at 5 sec (green), 10 sec (blue), and 15 sec (purple) after dithionite addition. Bottom three spectra: HydG∆CTD (100 mM) turnover of tyrosine (3 mM) with SAM (3 mM) initiated by addition of sodium dithionite and flash frozen in liquid propane at 5 sec (dark yellow), 15 sec (orange), and 30 sec (red) after dithionite addition. The EPR spectra parameters were microwave frequency 9.37 GHz; temperature 14 +/- 2K; power, 1 mW; time constant and conversion time, 20.48 ms; receiver gain, 1 x 104; modulation frequency, 100 kHz; and modulation amplitude, 10 G. 143 Figure 7.3. Broad field X-band EPR spectrum of HydG under turnover conditions. The g = 4.3 signal is from adventitiously bound high spin ferric iron. The relatively sharp feature centered at g = 2.0 is from the organic substrate based radical generated by HydG from tyrosine. The broad signals have previously been assigned to the [4Fe-4S]1+ clusters of HydG.(4) This spectrum is from the same sample used to generate the purple spectrum (WT 15 sec) in Figure 7.2 using EPR data collection parameters described in Figure 7.2. Substrate Promiscuity of HydG A recent X-ray crystal structure solution of the HydG homolog, NosL, gave new insight into the first step of the lyase activity for each of these enzymes. Previously, it 144 was proposed that tyrosine cleavage began by the abstraction of the phenolic hydrogen of tyrosine.(9) Interestingly, in the NosL structure substrate, tryptophan, was bound with its amine moiety positioned near the 5’-C of SAM where the deoxyadenosyl radical is generated upon SAM cleavage. This strongly suggested that the amino moiety of tryptophan would be the location of H-atom abstraction. Due to the strong similarity between NosL and HydG, one could infer that the site of H-atom abstraction by HydG is also the amino group of the substrate tyrosine. In order to examine this hypothesis with HydG, the rates of enzymatic production of 5’-deoxyadenosine and p-cresol were measured after a 30 min incubation of WT HydG with several tyrosine analogs in turnover assays (Figures 7.1 and 7.4). Figure 7.4. 5-Deoxyadenosine and p-cresol produced by HydG with substrate analogs. HydG (40 µM), SAM (1 mM), substrate analog (1 mM), and sodium dithionite (5 mM) were incubated at 37 ˚C for 30 min. The blue and red bars represent 5’-deoxyadenosine and p-cresol production, respectively. The error bars are +/- 1 standard deviation for injection replicates. 145 Most of these analogs retain the amino moiety but have alternate R-groups. WT HydG incubated with L-Tyr produced roughly 10 dAdo and p-cresol molecules per hour. When HydG was incubated with the analogs 4-fluoro-L-Phe, 4-amino-L-Phe, L-Phe, or L-Trp the production of dAdo was the same as the no-substrate controls (0.7 dAdo per hour). N-methyl-L-Tyr did stimulate dAdo production to 75% of L-Tyr but much of the SAM cleavage that occurred appeared to be uncoupled given that p-cresol production was only 25% that of L-Tyr. While O-methyl-L-Tyr appeared to increase SAM cleavage and produced p-cresol, this was later found to be due to L-Tyr contamination from the manufacturer (98% pure). Based on these results, the hydroxyl group on tyrosine appears to have a critical role in substrate specificity as well handling the radical intermediate during catalysis. If H-atom abstraction occurred with phenylalanine a benzylic radical would be produced. This radical would not have the hydrogen bond handle and would likely lead to side reactions and damage the enzyme. While two crystal structures of HydG have recently been published, neither have been solved with substrate in the active site.(8, 11) Given the high degree of conservation between amino acid sequences of NosL and HydG that comprise the core TIM barrels, the residues found in the active site of NosL structure could be used to determine those of HydG’s active site. The residue S338 (C. acetobutylicum HydG numbering) that corresponds to S340 (Streptomyces actuosus NosL numbering) appears to be involved in hydrogen bonding to the polar moieties of the R-groups of the substrates tyrosine and tryptophan, respectively. Through mutating this residue we hoped to reduce the substrate specificity of HydG previously described. The variant proteins S338A and S338V 146 removed the polar moiety that is believed to hydrogen bond to the hydroxyl group of tyrosine. By creation of a relatively hydrophobic pocket it was thought that the alternate substrate phenylalanine could bind and react in a manner similar to tyrosine in WT HydG. HydG S338D was made as the replacement of the serine with a negatively charged residue, which may allow histidine to occupy the active site in place of tyrosine. Interestingly, S338A and S338V variant proteins did not exhibit even low level unproductive SAM cleavage, as no appreciable dAdo was produced (data not shown). Also it was discovered that, S338D cleaved SAM producing dAdo at the low rate observed by the WT enzyme in the absence of substrate. The addition of tyrosine did not increase the rate of SAM cleavage with any of the variants as typical for WT enzyme, indicating that this residue is paramount for substrate recognition. EPR Spectroscopy of HydG-NTM Roach and coworkers recently proposed the auxiliary cluster of HydG is a [5Fe- 5S] cluster and suggested the fifth iron binds CO and CN- ligands produced by tyrosine lyase activity of HydG.(8) As evidence, they assigned a low field g = 4.3 centered signal to the [5Fe-5S] cluster (spectra in caption on lower left). They found that upon treatment with exogenous cyanide the g = 4.3 centered signal was abolished concomitant with the appearance of a S = ½ FeS cluster signal centered at g = 2, which they interpreted as the substitution of the fifth iron and sulfide by cyanide. On the other hand we observe a S = ½ FeS cluster signal centered at g = 2 that is changed upon the addition of exogenous cyanide but never observe the signal Roach and coworkers attributed to the [5Fe-5S] cluster. As with the turnover WT HydG spectra, the only signal in the g = 4.3 region is 147 from adventitiously bound iron and the signals reported by Roach and coworkers are not observed. Figure 7.5. EPR spectra of C. acetobutylicum HydG-NTM treated with cyanide. HydG (150 µM) reduced with sodium dithionite (10 mM)(top blue trace) and dithionite reduced with cyanide (3 mM)(bottom red trace). Roach and coworkers(8) (Figure S5) published the S. oneidensis HydG-NTM EPR spectra shown as insets in lower left. The EPR spectra parameters were microwave frequency 9.38 GHz; temperature 14 +/- 2K; power, 1 mW; time constant and conversion time, 40.96 ms; receiver gain, 1 x 104; modulation frequency, 100 kHz; and modulation amplitude, 10 G. Discussion The Tyrosine Lyase Active Site Multiple investigations have demonstrated that HydG uses radical SAM chemistry to cleave tyrosine and produce the carbon monoxide and cyanide ligands of the 148 H-cluster of [FeFe]-hydrogenase.(3, 5, 12, 13) Recently, multiple hypotheses, some conflicting, have been proposed regarding the mechanisms for tyrosine lyase activity, diatomic synthesis, and transfer of the diatomics to HydF.(8-10, 14, 15) Britt and coworkers observed a radical signal produced by S. oneidensis HydG during turnover with tyrosine that they assigned to a p-hydroxybenzylic radical.(10) This radical signal has been reproduced by our lab with the C. acetobutylicum HydG (Figures 7.2 and 7.3). Britt proposed that this radical was generated at the C-terminal cluster similar to MoaA(16) where the substrate binds to a [4Fe-4S] cluster positioning it for H-atom abstraction by the deoxyadenosyl radical produced near the N-terminal [4Fe-4S] cluster. Figure 7.6. A. Crystal Structure of Thermoanaerobacter italicus HydG (PDB ID: 4WCX) displaying the distance of 28.0 Å between the two [4Fe-4S] clusters. B. The active site of tryptophan lyase, NosL, (PDB ID: 4R33) contains the substrate tryptophan (carbons drawn in purple). The asterisk denotes where the deoxyadenosyl radical is generated and H-atom abstraction likely occurs. SAM and S-adenosylhomocysteine (SAH) are colored red. Color scheme unless otherwise noted: carbon (green), oxygen (red), nitrogen (blue), sulfur (yellow), and iron (orange). 149 Given the close alignment of HydG with other radical SAM enzymes with full TIM barrels (HydE, PylB, and BioB) we predicted (prior to the publication of the HydG crystal structure) that the location of the two [4Fe-4S] clusters and the distance between them (26 Å).(2, 17) We concluded that at this distance H-atom abstraction would not be possible if tyrosine were bound at the C-terminal cluster. The Roach lab (PDB ID: 4WCX) recently published a crystal structure solution in which the two [4Fe-4S] clusters (Figure 7.6A) are 28.0 Å apart, corroborating our conclusions drawn from the homology model investigations and refuting Britt’s supposition that tyrosine coordinates the C- terminal cluster. While neither group(8, 11) crystallized HydG with tyrosine in the active site, significant insight into this active site and the binding of the substrate has been garnered from the structural solution (PDB ID: 4R33) of the homolog NosL (Figure 7.6B).(9) The positioning of tryptophan, NosL’s substrate, relative to the location that the deoxyadenosyl radical is generated strongly suggests that H-atom abstraction occurs at the amine moiety rather than at the phenolic hydrogen of tyrosine, as previously suggested.(2, 10) To test this hypothesis a series of tyrosine analogs were screened in turnover assays for stimulation of SAM cleavage by HydG. The five analogs (Figures 7.1 and 7.4) that had the hydroxyl group removed from the phenyl group of tyrosine were unable to stimulate production of deoxyadenosine via SAM cleavage. Surprisingly, N- methyl-L-Tyr was still able to stimulate SAM cleavage albeit at a much lower rate than the natural substrate Tyr. However, ~60% of the SAM cleavage was off-pathway as production of p-cresol from tyrosine was not stoichiometric with dAdo production. Of all 150 the tyrosine analogs tested the only N-methyl-L-Tyr was able to stimulate 5’- deoxyadenosine production. This is likely because it retained the hydroxyl group that H- bonds to S338 aiding in orientation and recognition of the substrate in the substrate binding pocket. These findings in conjunction with the NosL crystal structure solution indicate that this active site is carefully tuned and that S338 is critical for substrate binding and cleavage. This new proposed mechanism (Figure 7.7) was further supported by the lack of SAM cleavage activity in the HydG variants S338A, S338V, and S338D. Figure 7.7. Proposed HydG tyrosine lyase mechanism. The close proximity of tyrosine’s amine in the active site near Arg320 would suggest the amine is deprotonated for catalysis. Figure 7.6B is the NosL structure that is used to model substrate binding in the HydG active site. Following H-atom abstraction from the amine position, the tyrosine radical intermediate undergoes Cα-Cβ bond cleavage producing dehydroglycine and the p-hydroxybenzylic radical. N CH C CH2 O O OH H H Arginine NH C H2N NH2 deoxyadenosyl N CH C CH2 O O OH H N CH C CH2 O O OH H 151 Characterization of the Auxiliary Cluster of HydG The crystal structure of HydG confirmed the presence of a complete (βα)8 TIM barrel containing a [4Fe-4S] cluster at the N-terminal portion of the protein and a separate domain at the C-terminal end that houses a second cluster.(8) The C-terminal cluster has been speculated to be a [5Fe-5S] moiety given its presence in only one of the subunits of the HydG dimer crystal structure and a low field EPR signal that was assigned to a S = 5/2 signal from this unique cluster (g = 9.5, 4.7, 4.1, and 3.7).(8) It should be noted that chemical reconstitution, as was performed on the protein that was crystalized by Roach and coworkers, can leaded to artifacts that appear in crystal structures.(18) The ambiguous coordination environment of the fifth iron including a sulfide bridge to the C-terminal cluster, a nonprotein derived free amino acid, two waters, and a histidinyl group make the assignment of the [5Fe-5S] cluster as being biologically relevant suspect. They also report that upon treatment of HydG with cyanide the low field signal disappears, which they speculate is due to the displacement of the fifth iron and sulfur with cyanide.(8) We feel this result is suspect for two main reasons. First, none of the broad field EPR spectra of WT and variant HydG collected by the Broderick lab have exhibited any low field spectral features other than a g = 4.3 junk iron signal. Given that the C. acetobutylicum enzyme used in the Broderick lab is active in producing 5’-deoxyadenosine, p-cresol, CO, and CN- as well as forming appreciable quantities of p-hydroxybenzylic radical, the presence of the low field signal reported by Britt and coworkers does not appear to correlate with active enzyme. Secondly, linear [3Fe-4S]+ clusters have a precedence in literature for having signals in the g = 4.3 range as originally mentioned by Britt and 152 coworkers in an earlier publication.(19, 20) Given these conflicting results amongst labs investigating HydG, future investigations are needed in elucidating the complex series of reactions occurring at HydG’s multiple active sites. The spectral features of HydG’s C- terminal cluster appear to be very different between research labs; whether or not Roach’s [5Fe-5S] cluster(8) is artifactual or mechanistically relevant is an important question and further research is required. Conclusions Prior to the publication of the HydG crystal structures we predicted Britt and coworkers(10) claim that tyrosine directly coordinated to the C-terminal cluster was unlikely. Using PylB and HydE as models, we found that the distance between HydG’s clusters placed tyrosine too far from the active site for H-atom abstraction.(2) Our mutagenesis studies and alternate substrate assays support the hypothesis that tyrosine’s the N-terminal active site of HydG is homologous to that of NosL. This strongly implies that tyrosine lyase activity is initiated by H-atom abstraction at the amine position of tyrosine leading to production of dehydroglycine and the p-hydroxybenzylic radical as we have hypothesized. The auxiliary cluster of HydG has recently been proposed to be a [5Fe-5S] cluster composed of a cubic [4Fe-4S] cluster with an additional iron ion bridged by a single sulfide, based on a crystal structure of exogenously reconstituted HydG and a low field EPR signal.(8) No HydG samples prepared in the Broderick lab exhibit such an EPR signal, despite being catalytically active, suggesting that whatever give rise to the low 153 field signal is not functionally relevant. While it is generally accepted that CO and CN- are derived from tyrosine by HydG’s radical SAM chemistry, a few important mechanistic details need to be resolved. The first key question is the mechanism of the diatomic ligands from the break down of the intermediate dehydroglycine. This question is intimately linked to the role of the C-terminal cluster. 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Elife, e03275. doi: 10.7554/eLife.03275 181 APPENDIX A SUPPORTING INFORMATION FOR CHAPTER 7 182 Figure S1. Primers used for Gibson assembly mutagenesis of HydG variants S338A, Q360E, and Y91F. 183 Figure S2. Gibson assembly cloning components and reaction conditions. A. Components for amplification of mutagenic fragments from WT template. B. Thermocycling conditions for amplification of mutagenic fragments. C. Gibson cloning assembly reagents. D. List of enzyme and reagents for Gibson assembly cocktail. E. Gibson assembly reaction components. A B C D E