Role of ammonia-lyases in the synthesis of the dithiomethylamine ligand during [FeFe]-hydrogenase maturation.

The generation of an active [FeFe]-hydrogenase requires the synthesis of a complex metal center, the H-cluster, by three dedicated maturases: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. A key step of [FeFe]-hydrogenase maturation is the synthesis of the dithiomethylamine (DTMA) bridging ligand, a process recently shown to involve the aminomethyl-lipoyl-H-protein from the glycine cleavage system, whose methylamine group originates from serine and ammonium. Here we use functional assays together with electron paramagnetic resonance and electron-nuclear double resonance spectroscopies to show that serine or aspartate together with their respective ammonia-lyase enzymes can provide the nitrogen for DTMA biosynthesis during in vitro [FeFe]-hydrogenase maturation. We also report bioinformatic analysis of the hyd operon, revealing a strong association with genes encoding ammonia-lyases, suggesting important biochemical and metabolic connections. Together, our results provide evidence that ammonia-lyases play an important role in [FeFe]-hydrogenase maturation by delivering the ammonium required for dithiomethylamine ligand synthesis.

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

ENDOR Spectroscopy Reveals the "Free" 5'-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe-4S]2+ Cluster.

1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.

Pyruvate formate-lyase activating enzyme: The catalytically active 5'-deoxyadenosyl radical caught in the act of H-atom abstraction.

Enzymes of the radical S-adenosyl-l-methionine (radical SAM, RS) superfamily, the largest in nature, catalyze remarkably diverse reactions initiated by H-atom abstraction. Glycyl radical enzyme activating enzymes (GRE-AEs) are a growing class of RS enzymes that generate the catalytically essential glycyl radical of GREs, which in turn catalyze essential reactions in anaerobic metabolism. Here, we probe the reaction of the GRE-AE pyruvate formate-lyase activating enzyme (PFL-AE) with the peptide substrate RVSG734YAV, which mimics the site of glycyl radical formation on the native substrate, pyruvate formate-lyase. Time-resolved freeze-quench electron paramagnetic resonance spectroscopy shows that at short mixing times reduced PFL-AE + SAM reacts with RVSG734YAV to form the central organometallic intermediate, Ω, in which the adenosyl 5'C is covalently bound to the unique iron of the [4Fe-4S] cluster. Freeze-trapping the reaction at longer times reveals the formation of the peptide G734• glycyl radical product. Of central importance, freeze-quenching at intermediate times reveals that the conversion of Ω to peptide glycyl radical is not concerted. Instead, homolysis of the Ω Fe-C5' bond generates the nominally "free" 5'-dAdo• radical, which is captured here by freeze-trapping. During cryoannealing at 77 K, the 5'-dAdo• directly abstracts an H-atom from the peptide to generate the G734• peptide radical trapped in the PFL-AE active site. These observations reveal the 5'-dAdo• radical to be a well-defined intermediate, caught in the act of substrate H-atom abstraction, providing new insights into the mechanistic steps of radical initiation by RS enzymes.

Computational Description of Alkylated Iron-Sulfur Organometallic Clusters.

The radical S-adenosyl methionine (SAM) enzyme superfamily has widespread roles in hydrogen atom abstraction reactions of crucial biological importance. In these enzymes, reductive cleavage of SAM bound to a [4Fe-4S]1+ cluster generates the 5'-deoxyadenosyl radical (5'-dAdo•) which ultimately abstracts an H atom from the substrate. However, overwhelming experimental evidence has surprisingly revealed an obligatory organometallic intermediate Ω exhibiting an Fe-C5'-adenosyl bond, whose properties are the target of this theoretical investigation. We report a readily applied, two-configuration version of broken symmetry DFT, denoted 2C-DFT, designed to allow the accurate description of the hyperfine coupling constants and g-tensors of an alkyl group bound to a multimetallic iron-sulfur cluster. This approach has been validated by the excellent agreement of its results both with those of multiconfigurational complete active space self-consistent field computations for a series of model complexes and with the results from electron nuclear double-resonance/electron paramagnetic resonance spectroscopic studies for the crystallographically characterized complex, M-CH3, a [4Fe-4S] cluster with a Fe-CH3 bond. The likewise excellent agreement between spectroscopic results and 2C-DFT computations for Ω confirm its identity as an organometallic complex with a bond between an Fe of the [4Fe-4S] cluster and C5' of the deoxyadenosyl moiety, as first proposed.

Mechanism of Radical Initiation in the Radical SAM Enzyme Superfamily.

Radical S-adenosylmethionine (SAM) enzymes use a site-differentiated [4Fe-4S] cluster and SAM to initiate radical reactions through liberation of the 5'-deoxyadenosyl (5'-dAdo•) radical. They form the largest enzyme superfamily, with more than 700,000 unique sequences currently, and their numbers continue to grow as a result of ongoing bioinformatics efforts. The range of extremely diverse, highly regio- and stereo-specific reactions known to be catalyzed by radical SAM superfamily members is remarkable. The common mechanism of radical initiation in the radical SAM superfamily is the focus of this review. Most surprising is the presence of an organometallic intermediate, Ω, exhibiting an Fe-C5'-adenosyl bond. Regioselective reductive cleavage of the SAM S-C5' bond produces 5'-dAdo• to form Ω, with the regioselectivity originating in the Jahn-Teller effect. Ω liberates the free 5'-dAdo• as the catalytically active intermediate through homolysis of the Fe-C5' bond, in analogy to Co-C5' bond homolysis in B12, which was once viewed as biology's choice of radical generator.

The Fe Protein Cycle Associated with Nitrogenase Catalysis Requires the Hydrolysis of Two ATP for Each Single Electron Transfer Event.

A central feature of the current understanding of dinitrogen (N2) reduction by the enzyme nitrogenase is the proposed coupling of the hydrolysis of two ATP, forming two ADP and two Pi, to the transfer of one electron from the Fe protein component to the MoFe protein component, where substrates are reduced. A redox-active [4Fe-4S] cluster associated with the Fe protein is the agent of electron delivery, and it is well known to have a capacity to cycle between a one-electron-reduced [4Fe-4S]1+ state and an oxidized [4Fe-4S]2+ state. Recently, however, it has been shown that certain reducing agents can be used to further reduce the Fe protein [4Fe-4S] cluster to a super-reduced, all-ferrous [4Fe-4S]0 state that can be either diamagnetic (S = 0) or paramagnetic (S = 4). It has been proposed that the super-reduced state might fundamentally alter the existing model for nitrogenase energy utilization by the transfer of two electrons per Fe protein cycle linked to hydrolysis of only two ATP molecules. Here, we measure the number of ATP consumed for each electron transfer under steady-state catalysis while the Fe protein cluster is in the [4Fe-4S]1+ state and when it is in the [4Fe-4S]0 state. Both oxidation states of the Fe protein are found to operate by hydrolyzing two ATP for each single-electron transfer event. Thus, regardless of its initial redox state, the Fe protein transfers only one electron at a time to the MoFe protein in a process that requires the hydrolysis of two ATP.

Connecting the geometric and electronic structures of the nitrogenase iron-molybdenum cofactor through site-selective 57Fe labelling.

Understanding the chemical bonding in the catalytic cofactor of the Mo nitrogenase (FeMo-co) is foundational for building a mechanistic picture of biological nitrogen fixation. A persistent obstacle towards this goal has been that the 57Fe-based spectroscopic data-although rich with information-combines responses from all seven Fe sites, and it has therefore not been possible to map individual spectroscopic responses to specific sites in the three-dimensional structure. Here we have addressed this challenge by incorporating 57Fe into a single site of FeMo-co. Spectroscopic analysis of the resting state informed on the local electronic structure of the terminal Fe1 site, including its oxidation state and spin orientation, and, in turn, on the spin-coupling scheme for the entire cluster. The oxidized resting state and the first intermediate in nitrogen fixation were also characterized, and comparisons with the resting state provided molecular-level insights into the redox chemistry of FeMo-co.

Radical SAM enzymes: Nature's choice for radical reactions.

Enzymes that use a [4Fe-4S]1+ cluster plus S-adenosyl-l-methionine (SAM) to initiate radical reactions (radical SAM) form the largest enzyme superfamily, with over half a million members across the tree of life. This review summarizes recent work revealing the radical SAM reaction pathway, which ultimately liberates the 5'-deoxyadenosyl (5'-dAdo•) radical to perform extremely diverse, highly regio- and stereo-specific, transformations. Most surprising was the discovery of an organometallic intermediate Ω exhibiting an Fe-C5'-adenosyl bond. Ω liberates 5'-dAdo• through homolysis of the Fe-C5' bond, in analogy to Co-C5' bond homolysis in B12 , previously viewed as biology's paradigmatic radical generator. The 5'-dAdo• has been trapped and characterized in radical SAM enzymes via a recently discovered photoreactivity of the [4Fe-4S]+ /SAM complex, and has been confirmed as a catalytically active intermediate in enzyme catalysis. The regioselective SAM S-C bond cleavage to produce 5'-dAdo• originates in the Jahn-Teller effect. The simplicity of SAM as a radical precursor, and the exquisite control of 5'-dAdo• reactivity in radical SAM enzymes, may be why radical SAM enzymes pervade the tree of life, while B12 enzymes are only a few.

Effects of Desiccation and Freezing on Microbial Ionizing Radiation Survivability: Considerations for Mars Sample Return.

Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.

Characterization by ENDOR Spectroscopy of the Iron-Alkyl Bond in a Synthetic Counterpart of Organometallic Intermediates in Radical SAM Enzymes.

Members of the radical S-adenosyl-l-methionine (SAM) enzyme superfamily initiate a broad spectrum of radical transformations through reductive cleavage of SAM by a [4Fe-4S]1+ cluster it coordinates to generate the reactive 5'-deoxyadenosyl radical (5'-dAdo•). However, 5'-dAdo• is not directly liberated for reaction and instead binds to the unique Fe of the cluster to create the catalytically competent S = 1/2 organometallic intermediate Ω. An alternative mode of reductive SAM cleavage, especially seen photochemically, instead liberates CH3•, which forms the analogous S = 1/2 organometallic intermediate with an Fe-CH3 bond, ΩM. The presence of a covalent Fe-C bond in both structures was established by the ENDOR observation of 13C and 1H hyperfine couplings to the alkyl groups that show isotropic components indicative of Fe-C bond covalency. The synthetic [Fe4S4]3+-CH3 cluster, M-CH3, is a crystallographically characterized analogue to ΩM that exhibits the same [Fe4S4]3+ cluster state as Ω and ΩM, and thus an analysis of its spectroscopic properties─and comparison with those of Ω and ΩM─can be grounded in its crystal structure. We report cryogenic (2 K) EPR and 13C/1/2H ENDOR measurements on isotopically labeled M-CH3. At low temperatures, the complex exhibits EPR spectra from two distinct conformers/subpopulations. ENDOR shows that at 2 K, one contains a static methyl, but in the other, the methyl undergoes rapid tunneling/hopping rotation about the Fe-CH3 bond. This generates an averaged hyperfine coupling tensor whose analysis requires an extended treatment of rotational averaging. The methyl group 13C/1/2H hyperfine couplings are compared with the corresponding values for Ω and ΩM.

13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing "Heart of Steel".

Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe-9S-C-Mo-homocitrate] contained within the MoFe protein α-subunit. The cofactor contains a CFe6 core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted 13C labeling of the carbon enables electron-nuclear double resonance (ENDOR) spectroscopy to sensitively monitor the electronic properties of the Fe-C bonds and the spin-coupling scheme adopted by the FeMo-cofactor metal ions. This report compares 13CFe6 ENDOR measurements for (i) the wild-type protein resting state (E0; α-Val70) to those of (ii) α-Ile70, (iii) α-Ala70-substituted proteins; (iv) crystallographically characterized CO-inhibited "hi-CO" state; (v) E4(4H) Janus intermediate, activated for N2 binding/reduction by accumulation of 4[e-/H+]; (vi) E4(2H)* state containing a doubly reduced FeMo-cofactor without Fe-bound substrates; and (vii) propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, both S = 1/2 and 3/2 exhibited near-zero 13C isotropic hyperfine coupling constants, Ca = [-1.3 ↔ +2.7] MHz. Density functional theory computations and natural bond orbital analysis of the Fe-C bonds show that this occurs because a (3 spin-up/3 spin-down) spin-exchange configuration of CFe6 Fe-ion spins produces cancellation of large spin-transfers to carbon in each Fe-C bond. Previous X-ray diffraction and DFT both indicate that trigonal-prismatic geometry around carbon is maintained with high precision in all these states. The persistent structure and Fe-C bonding of the CFe6 core indicate that it does not provide a functionally dynamic (hemilabile) "beating heart"─instead it acts as "a heart of steel", stabilizing the structure of the FeMo-cofactor-active site during nitrogenase catalysis.

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis.

SignificanceMethanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.

[FeFe]-Hydrogenase: Defined Lysate-Free Maturation Reveals a Key Role for Lipoyl-H-Protein in DTMA Ligand Biosynthesis.

Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN- , and dithiomethylamine (DTMA)-coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.

Mechanism of Radical S-Adenosyl-l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5'-Deoxyadenosyl Radical.

Radical S-adenosyl-l-methionine (SAM) enzymes employ a [4Fe-4S] cluster and SAM to initiate diverse radical reactions via either H-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR) spectroscopy to provide snapshots of the reaction pathway in an adenosylation reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme on a peptide substrate containing a dehydroalanine residue in place of the target glycine. The reaction proceeds via the initial formation of the organometallic intermediate Ω, as evidenced by the characteristic EPR signal with g∥ = 2.035 and g⊥ = 2.004 observed when the reaction is freeze-quenched at 500 ms. Thermal annealing of frozen Ω converts it into a second paramagnetic species centered at giso = 2.004; this second species was generated directly using freeze-quench at intermediate times (∼8 s) and unequivocally identified via isotopic labeling and EPR spectroscopy as the tertiary peptide radical resulting from adenosylation of the peptide substrate. An additional paramagnetic species observed in samples quenched at intermediate times was revealed through thermal annealing while frozen and spectral subtraction as the SAM-derived 5'-deoxyadenosyl radical (5'-dAdo•). The time course of the 5'-dAdo• and tertiary peptide radical EPR signals reveals that the former generates the latter. These results thus support a mechanism in which Ω liberates 5'-dAdo• by Fe-C5' bond homolysis, and the 5'-dAdo• attacks the dehydroalanine residue of the peptide substrate to form the adenosylated peptide radical species. The results thus provide a picture of a catalytically competent 5'-dAdo• intermediate trapped just prior to reaction with the substrate.

A New Reaction for Improved Calibration of EPR Rapid-Freeze Quench Times: Kinetics of Ethylene Diamine Tetraacetate (EDTA) Transfer from Calcium(II) to Copper(II).

The kinetics of the transfer of the chelate, ethylenediamine tetraacetate (EDTA), from Calcium(II) to Copper(II) in imidazole (Im) buffers near neutral pH, corresponding to the conversion, [Cu(II)Im4]2+→ [Cu(II)EDTA]2-, are characterized with stopped-flow absorption spectroscopy and implemented as a tool for calibrating the interval between mixing and freezing, the freeze-quench time (t Q ), of a rapid freeze-quench (RFQ) apparatus. The kinetics of this reaction are characterized by monitoring changes in UV-visible spectra (300 nm) due to changes in the charge-transfer band associated with the Cu2+ ions upon EDTA binding. Stopped-flow measurements show that the rates of conversion of the Cu2+ ions exhibit exponential kinetics on millisecond time scales at pH values less than 6.8. In parallel, we have developed a simple but precise method to quantitate the speciation of frozen solution mixtures of [Cu(II)(EDTA)]2- and tetraimidazole Cu(II) ([Cu(Im)4]2+) in X-band EPR spectra. The results are implemented in a simple high-precision 'recipe' for determining t Q . These procedures are more accurate and precise than the venerable reaction of aquometmyoglobin with azide for calibrating RFQ apparatus, with the benefit of avoiding high-concentrations of toxic azide solutions.

Hydrocarbon Oxidation by an Exposed, Multiply Bonded Iron(III) Oxo Complex.

The iron oxo unit, [Fe=O] n+ is a critical intermediate in biological oxidation reactions. While its higher oxidation states are well studied, relatively little is known about the least-oxidized form [FeIII=O]+. Here, the thermally stable complex PhB(AdIm)3Fe=O has been structurally, spectroscopically, and computationally characterized as a bona fide iron(III) oxo. An unusually short Fe-O bond length is consistent with iron-oxygen multiple bond character and is supported by electronic structure calculations. The complex is thermally stable yet is able to perform hydrocarbon oxidations, facilitating both C-O bond formation and dehydrogenation reactions.

S-Adenosyl-l-ethionine is a Catalytically Competent Analog of S-Adenosyl-l-methione (SAM) in the Radical SAM Enzyme HydG.

Radical S-adenosyl-l-methionine (SAM) enzymes initiate biological radical reactions with the 5'-deoxyadenosyl radical (5'-dAdo•). A [4Fe-4S]+ cluster reductively cleaves SAM to form the Ω organometallic intermediate in which the 5'-deoxyadenosyl moiety is directly bound to the unique iron of the [4Fe-4S] cluster, with subsequent liberation of 5'-dAdo•. Here we present synthesis of the SAM analog S-adenosyl-l-ethionine (SAE) and show SAE is a mechanistically-equivalent SAM-alternative for HydG, both supporting enzymatic turnover of substrate tyrosine and forming the organometallic intermediate Ω. Photolysis of SAE bound to HydG forms an ethyl radical trapped in the active site. The ethyl radical withstands prolonged storage at 77 K and its EPR signal is only partially lost upon annealing at 100 K, making it significantly less reactive than the methyl radical formed by SAM photolysis. Upon annealing above 77K, the ethyl radical adds to the [4Fe-4S]2+ cluster, generating an ethyl-[4Fe-4S]3+ organometallic species termed ΩE.

Active-Site Controlled, Jahn-Teller Enabled Regioselectivity in Reductive S-C Bond Cleavage of S-Adenosylmethionine in Radical SAM Enzymes.

Catalysis by canonical radical S-adenosyl-l-methionine (SAM) enzymes involves electron transfer (ET) from [4Fe-4S]+ to SAM, generating an R3S0 radical that undergoes regioselective homolytic reductive cleavage of the S-C5' bond to generate the 5'-dAdo· radical. However, cryogenic photoinduced S-C bond cleavage has regioselectively yielded either 5'-dAdo· or ·CH3, and indeed, each of the three SAM S-C bonds can be regioselectively cleaved in an RS enzyme. This diversity highlights a longstanding central question: what controls regioselective homolytic S-C bond cleavage upon SAM reduction? We here provide an unexpected answer, founded on our observation that photoinduced S-C bond cleavage in multiple canonical RS enzymes reveals two enzyme classes: in one, photolysis forms 5'-dAdo·, and in another it forms ·CH3. The identity of the cleaved S-C bond correlates with SAM ribose conformation but not with positioning and orientation of the sulfonium center relative to the [4Fe-4S] cluster. We have recognized the reduced-SAM R3S0 radical is a (2E) state with its antibonding unpaired electron in an orbital doublet, which renders R3S0 Jahn-Teller (JT)-active and therefore subject to vibronically induced distortion. Active-site forces induce a JT distortion that localizes the odd electron in a single priority S-C antibond, which undergoes regioselective cleavage. In photolytic cleavage those forces act through control of the ribose conformation and are transmitted to the sulfur via the S-C5' bond, but during catalysis thermally induced conformational changes that enable ET from a cluster iron generate dominant additional forces that specifically select S-C5' for cleavage. This motion also can explain how 5'-dAdo· subsequently forms the organometallic intermediate Ω.

Radical SAM Enzyme Spore Photoproduct Lyase: Properties of the Ω Organometallic Intermediate and Identification of Stable Protein Radicals Formed during Substrate-Free Turnover.

Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]1+ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S-C5' bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5' of the 5'-deoxyadenosyl radical (5'-dAdo•). During catalysis, homolytic cleavage of the Fe-C5' bond liberates 5'-dAdo• for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze-quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe-C5' bond of τ ≈ 5-6 s, likely shortened by enzymatic activation as is the case with the Co-C5' bond of B12. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5'-dAdo• radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile• or Cys• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.