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.

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.

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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 (2 E) 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.

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