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.

Radical SAM (RS) enzymes use S-adenosyl-L-methionine (SAM) and a [4Fe-4S] cluster to initiate a broad spectrum of radical transformations throughout all kingdoms of life. We report here that low-temperature photoinduced electron transfer from the [4Fe-4S] 1+ cluster to bound SAM in the active site of the hydrogenase maturase RS enzyme, HydG, results in specific homolytic cleavage of the S-CH 3 bond of SAM, rather than the S-C5′ bond as in the enzymecatalyzed (thermal) HydG reaction. This result is in stark contrast to a recent report in which photoinduced ET in the RS enzyme pyruvate formate-lyase activating enzyme cleaved the S-C5′ bond to generate a 5′-deoxyadenosyl radical, and provides the first direct evidence for homolytic S-CH 3 bond cleavage in a RS enzyme. Photoinduced ET in HydG generates a trapped • CH 3 radical, as well as a small population of an organometallic species with an Fe-CH 3 bond, denoted Ω M. The • CH 3 radical is surprisingly found to exhibit rotational diffusion in the HydG active site at temperatures as low as 40 K, and is rapidly quenched: whereas 5′-dAdo • is stable indefinitely at 77 K, • CH 3 quenches with a half-time of ~2 min at this temperature. The rapid quenching and rotational/translational freedom of • CH 3 shows that enzymes would be unable to harness this radical as a regio-and stereospecific H atom abstractor during catalysis, in contrast to the exquisite control achieved with the enzymatically generated 5′-dAdo • .

Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN À , 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.

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