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 synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-L-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN − ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN − and the CO precursor − :CO 2 H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second ironsulfur cluster coordination by measuring the in vitro production of CO, CN − , and − :CO 2 H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN − , and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO) x (CN) y transfer to the HydF carrier/scaffold. (4) showed that the active site is composed of a conventional [4Fe-4S] cubane connected by a cysteine thiolate to a binuclear FeFe unit, in which each iron ion is terminally coordinated by one CN − ligand and one CO ligand and by a third CO molecule that bridges the two metals (5). Unexpectedly, we also found that a small molecule first postulated (6), and now indirectly confirmed (7), to be dithiomethylamine (DTMA) bridges the two Fe ions (Fig. 1).The [4Fe-4S] cubane bridged to the binuclear [FeFe] unit has been collectively called the H-cluster (1). Work from several laboratories has shown that the maturation of the [FeFe] center requires at least three protein maturases: HydF that has GTPase activity and appears to be both a [FeFe] center scaffold and carrier (8, 9), HydG that synthesizes CO and CN − from tyrosine (10-13), and HydE that, by elimination, should be involved in the synthesis of the DTMA bridge (14, 15). Both HydE and HydG are members of the large radical S-adenosyl-L-methionine (SAM) protein family (16,17). With the recent reports of HydG crystal structures from Carboxydothermus hydrogenoformans (Ch) by us (18) and from Thermoanaerobacter italicus (Ti) by Dinis et al. (19), X-ray models are now available for the three maturases (20, 21); however, unambiguous structure-function relationships have been proposed only in the case of HydG. Indeed, site-directed mutational studies have shown that CO and CN − syntheses are affected by either the deletion of the maturase C-terminal region, where a second iron-sulfur cluster binds (22), or Cys-to-Ser mutations in its corresponding CxxCx 22 C binding motif (10, 13). In addition, it has been shown that HydG synthesizes Fe(CO) x (CN) y precursors (x = 1 or 2; y = 1) of the [FeFe] catalytic unit (23). The two HydG crystal structures are very similar at the SAM and [4Fe-4S] cluster-containing (β/α) 8 TIM-like barrel, common to several radical SAM proteins (16) (Fig. 2). Conversely, there are significant differences in the composition of the extra C-terminal second (s) iron-sulfur cluster. In our crystals, ChHydG lacks th...