The B12-independent glycerol dehydratase activating enzyme from Clostridium butyricum cleaves SAM to produce 5'-deoxyadenosine and not 5'-deoxy-5'-(methylthio)adenosine.

Glycerol dehydratase activating enzyme (GD-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential amino acid backbone radical onto glycerol dehydratase in bacteria under anaerobic conditions. Although GD-AE is closely homologous to other radical SAM activases that have been shown to cleave the S-C(5') bond of SAM to produce 5'-deoxyadenosine (5'-dAdoH) and methionine, GD-AE from Clostridium butyricum has been reported to instead cleave the S-C(γ) bond of SAM to yield 5'-deoxy-5'-(methylthio)adenosine (MTA). Here we re-investigate the SAM cleavage reaction catalyzed by GD-AE and show that it produces the widely observed 5'-dAdoH, and not the less conventional product MTA.

HydG, the "dangler" iron, and catalytic production of free CO and CN-: implications for [FeFe]-hydrogenase maturation.

The organometallic H-cluster of the [FeFe]-hydrogenase consists of a [4Fe-4S] cubane bridged via a cysteinyl thiolate to a 2Fe subcluster ([2Fe]H) containing CO, CN-, and dithiomethylamine (DTMA) ligands. The H-cluster is synthesized by three dedicated maturation proteins: the radical SAM enzymes HydE and HydG synthesize the non-protein ligands, while the GTPase HydF serves as a scaffold for assembly of [2Fe]H prior to its delivery to the [FeFe]-hydrogenase containing the [4Fe-4S] cubane. HydG uses l-tyrosine as a substrate, cleaving it to produce p-cresol as well as the CO and CN- ligands to the H-cluster, although there is some question as to whether these are formed as free diatomics or as part of a [Fe(CO)2(CN)] synthon. Here we show that Clostridium acetobutylicum (C.a.) HydG catalyzes formation of multiple equivalents of free CO at rates comparable to those for CN- formation. Free CN- is also formed in excess molar equivalents over protein. A g = 8.9 EPR signal is observed for C.a. HydG reconstituted to load the 5th "dangler" iron of the auxiliary [4Fe-4S][FeCys] cluster and is assigned to this "dangler-loaded" cluster state. Free CO and CN- formation and the degree of activation of [FeFe]-hydrogenase all occur regardless of dangler loading, but are increased 10-35% in the dangler-loaded HydG; this indicates the dangler iron is not essential to this process but may affect relevant catalysis. During HydG turnover in the presence of myoglobin, the g = 8.9 signal remains unchanged, indicating that a [Fe(CO)2(CN)(Cys)] synthon is not formed at the dangler iron. Mutation of the only protein ligand to the dangler iron, H272, to alanine nearly completely abolishes both free CO formation and hydrogenase activation, however results show this is not due solely to the loss of the dangler iron. In experiments with wild type and H272A HydG, and with different degrees of dangler loading, we observe a consistent correlation between free CO/CN- formation and hydrogenase activation. Taken in full, our results point to free CO/CN-, but not an [Fe(CO)2(CN)(Cys)] synthon, as essential species in hydrogenase maturation.

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.

The Elusive 5'-Deoxyadenosyl Radical: Captured and Characterized by Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopies.

The 5'-deoxyadenosyl radical (5'-dAdo·) abstracts a substrate H atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, 5'-dAdo· has eluded characterization despite efforts spanning more than a half-century. Here, we report generation of 5'-dAdo· in a RS enzyme active site at 12 K using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe-4S]+ cluster to the coordinated S-adenosylmethionine (SAM) to induce homolytic S-C5' bond cleavage. We unequivocally reveal the structure of this long-sought radical species through the use of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies with isotopic labeling, complemented by density-functional computations: a planar C5' (2pπ) radical (∼70% spin occupancy); the C5'(H)2 plane is rotated by ∼37° (experiment)/39° (DFT) relative to the C5'-C4'-(C4'-H) plane, placing a C5'-H antiperiplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4'-H. The agreement between φ from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo· itself, and not determined by its environment.

Mechanistic Studies of Radical SAM Enzymes: Pyruvate Formate-Lyase Activating Enzyme and Lysine 2,3-Aminomutase Case Studies.

The radical SAM enzyme superfamily is large and diverse, with ever-increasing numbers of examples of characterized reactions. This chapter focuses on the methodology we have developed over the last 25 years for working with these enzymes, with the specific examples discussed being the pyruvate formate-lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM). Both enzymes are purified from overexpressing Escherichia coli, but differ in that PFL-AE is expressed without an affinity tag and does not require iron-sulfur cluster reconstitution, while LAM purification is carried out through use of a His6 affinity tag and the enzyme benefits from cluster reconstitution. Because of radical SAM enzymes' catalytic need for a [4Fe-4S] cluster, we present methods for characterization and incorporation of a full [4Fe-4S] cluster in addition to enzyme activity assay protocols. Synthesis of SAM (S-adenosyl-l-methionine) and its analogs have played an important role in our mechanistic studies of radical SAM enzymes, and their synthetic methods are also presented in detail.

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis.

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5'-deoxyadenosyl radical (5'-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe-C5' bond between 5'-dAdo· and the [4Fe-4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5'-dAdo· intermediate. Liberation of a reactive 5'-dAdo· intermediate via homolytic metal-carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.