Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN − ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO) x (CN) y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα) 8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ 2 -sulfide ion to a mononuclear Fe 2+ center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe (CO) x (CN) y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.radical SAM enzyme | tyrosine lyase | H-cluster biosynthesis T he assembly of the [FeFe]-hydrogenase diiron subcluster (1, 2) requires three maturase proteins, HydE, HydF, and HydG (3), and in vitro, they can assemble an active hydrogenase (4). The sequence and structure of the maturase HydE (5) indicates that it is a member of the radical S-adenosyl methionine (SAM) superfamily, although the biochemical function of HydE has not been experimentally determined. The GTPase HydF (6, 7) has been shown to transfer synthetic (8) or biologically derived (7, 9) diiron subclusters into apo-hydrogenase, suggesting that HydF functions as a template for diiron subcluster assembly. The tyrosine lyase HydG is also a member of the radical SAM superfamily and uses SAM and a reductant (such as dithionite) to cleave the Cα-Cβ bond of tyrosine, yielding p-cresol as the side chain-derived byproduct (10) and fragmenting the amino acid moiety into cyanide (CN − ) (11) and carbon monoxide (CO) (12), which are ultimately incorporated as ligands in the H cluster of the [FeFe]-hydrogenase HydA (4). Two site-differentiated [4Fe-4S] clusters in HydG have been identified using a combination of spectroscopy and site-directed mutagenesis (12-16). The cluster bound close to the N terminus ([4Fe-4S] RS ) by the CX 3 CX 2 C cysteine triad motif (SI Appendix, Fig. S1) is typical of the radical SAM superfamily (17, 18) and has been shown to catalyze the reductive cl...

Hydrogenases catalyze the redox interconversion of protons and H 2 , an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO) 2 (CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe 2+ binding and synthon release.FeFe hydrogenase | metallocofactor biosynthesis | HydG F eFe hydrogenases catalyze the reversible interconversion of H 2 with protons and electrons, and thereby provide either an electron source or an electron sink for a variety of metabolic processes (1). Hydrogenase reactivity occurs at the H cluster, which consists of a conventional [4Fe-4S] H subcluster coupled to an organometallic [2Fe] H subcluster that features a 2-aza-1,3-propanedithiolate ("azadithiolate") ligand and multiple CO and CN -ligands (Fig. 1A) (2, 3). The biosynthesis of the H cluster has garnered much attention (4, 5) given its unusual structure and exceptional H 2 production activity (6). Whereas the [4Fe-4S] H subcluster is inserted by the housekeeping Fe-S cluster machinery, the [2Fe] H subcluster is synthesized and inserted by three accessory proteins: the HydE, HydF, and HydG maturases (5, 7-9). Both L-tyrosine (Tyr) and L-cysteine (Cys) have been shown to stimulate in vitro [2Fe] H subcluster biosynthesis (10, 11) with Tyr serving as the precursor to the CO and CN -ligands (12-14); the role of Cys in H-cluster maturation is less clear and an emerging area of focus (15).Significant progress has been made toward elucidating the individual functions of the maturases (5). HydG is a member of the radical S-adenosyl-L-methionine (SAM) family of enzymes (16) (Fig. 1B). The substrate and product of the radical SAM enzyme HydE are presently unknown, although it is thought that HydE plays a role in building the azadithiolate ligand (5, 15). HydG and HydE are thought to function in concert with the GTP-hydrolyzing enzyme HydF (18, 19) to generate a [2Fe] H subcluster-like precursor (20-22) that is transferred to the hydrogenase apoprotein (apo-HydA) to yield the mature H cluster. This mechanistic framework continues to undergo substantial refinement as the chemical details of these processes are unraveled.HydG contains two Fe-S clusters that play separate roles in building the [Fe(CO) 2 (CN)] synthon (17,(23)(24)(25)(26). Cleavage of Tyr to CO and CN -is initiated at the N-terminal, SAM-binding [4Fe-4S] RS cluster where one-electron reduction of SAM generates the 5′-deoxyadenosyl radical (5′-dAdo•) (Fig. 1B). Subsequent H-atom abstraction from the amino group (27) of Tyr (28) induces Cα-Cβ bond cleavage. The resulting 4-hydroxybenzyl radical (4HOB•) has been observed by EP...

Hydrogenase enzymes catalyze the rapid and reversible interconversion of H2 with protons and electrons. The active site of the [FeFe] hydrogenase is the H cluster, which consists of a [4Fe–4S]H subcluster linked to an organometallic [2Fe]H subcluster. Understanding the biosynthesis and catalytic mechanism of this structurally unusual active site will aid in the development of synthetic and biological hydrogenase catalysts for applications in solar fuel generation. The [2Fe]H subcluster is synthesized and inserted by three maturase enzymes—HydE, HydF, and HydG—in a complex process that involves inorganic, organometallic, and organic radical chemistry. HydG is a member of the radical S-adenosyl-L-methionine (SAM) family of enzymes and is thought to play a prominent role in [2Fe]H subcluster biosynthesis by converting inorganic Fe2+, L-cysteine (Cys), and L-tyrosine (Tyr) into an organometallic [(Cys)Fe(CO)2(CN)]− intermediate that is eventually incorporated into the [2Fe]H subcluster. In this Forum Article, the mechanism of [2Fe]H subcluster biosynthesis is discussed with a focus on how this key [(Cys)Fe(CO)2(CN)]− species is formed. Particular attention is given to the initial metallocluster composition of HydG, the modes of substrate binding (Fe2+, Cys, Tyr, and SAM), the mechanism of SAM-mediated Tyr cleavage to CO and CN−, and the identification of the final organometallic products of the reaction.

An in situ Raman method was developed to characterize the disproportionation of two salts involving a complex polymorphic landscape comprising up to two metastable and one stable freebase forms. Few precedents exist for Raman calibration procedures for solid form quantitation involving more than two polymorphs, while no literature examples were found for cases with multiple metastable forms. Therefore, a new Raman calibration procedure was proposed by directly using disproportionation experiments to generate multiple calibration samples encompassing a range of polymorph ratios through in-line Raman measurements complemented by off-line reference X-ray diffraction measurements. The developed Raman methods were capable of accurately quantitating each solid form in situ when solid concentration variation was incorporated into the calibration dataset. The kinetic understanding of the thermodynamically driven polymorphic conversions gained from this Raman method guided the selection of the salt best suited for the delivery of the active ingredient in the drug product. This work provided a spectroscopic and mathematical approach for simultaneously quantitating multiple polymorphs from a complex mixture of solids with the objective of real-time monitoring.