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...

What's your poison? Hydrogenases catalyze the reversible formation of dihydrogen from two electrons and two protons. The maturation of the [FeFe]‐hydrogenase active‐site cofactor (H cluster) requires three gene products, HydE, HydF, and HydG. Cyanide has been characterized as one of the products of tyrosine cleavage by the S‐adenosylmethionine‐dependent enzyme HydG, clarifying its role in H‐cluster biosynthesis. DOA=deoxyadenosine.

The radical S-adenosyl-L-methionine (AdoMet) enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase H-cluster assembly. It catalyzes L-tyrosine cleavage to yield the H-cluster cyanide and carbon monoxide ligands as well as p-cresol. Clostridium acetobutylicum HydG contains the conserved CX3CX2C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX2CX22C motif proposed to coordinate a second [4Fe-4S] cluster. To improve our understanding of the roles of each of these iron-sulfur clusters in catalysis, we have generated HydG variants lacking either the N- or C-terminal cluster and examined these using spectroscopic and kinetic methods. We have used iron analyses, UV-visible spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant that cannot coordinate the radical AdoMet cluster to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (ΔCTD) harboring only the N-terminal cluster demonstrates that both clusters have similar UV-visible and EPR spectral properties, but that AdoMet binding and cleavage occur only at the N-terminal radical AdoMet cluster. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, we compared the Michaelis-Menten constants for AdoMet and L-tyrosine for reconstituted wild-type, C386S, and ΔCTD HydG and demonstrate that these C-terminal modifications do not affect the affinity for AdoMet but that the affinity for L-tyrosine is drastically reduced compared to that of wild-type HydG. Further detailed kinetic characterization of these HydG mutants demonstrates that the C-terminal cluster and residues are not essential for L-tyrosine cleavage to p-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO.

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Hydrogenases catalyze the reversible reduction of protons to yield molecular hydrogen (H 2 ) and occur in three evolutionarily unrelated forms termed the [Fe]-, [FeFe]-, and [NiFe]hydrogenases. [1,2] [FeFe]-hydrogenases all contain a complex active-site cofactor termed the H cluster (1, Scheme 1) that consists of a regular [4Fe4S] cluster bridged by a shared cysteine thiolate sulfur atom to a 2Fe subcluster with biologically unique carbon monoxide, cyanide, and dithiolate ligands. [3,4] The H cluster is biosynthesized in a stepwise process in which generalized host cell machinery [5] is directed towards the synthesis of a [4Fe4S] subcluster with subsequent synthesis and insertion of the 2Fe subcluster by specialized hyd encoded proteins, HydE, HydF, and HydG. [1,6] HydE and HydG are radical S-adenosylmethionine (AdoMet) enzymes thought to be responsible for the synthesis and proper incorporation of the nonprotein ligands. [1,[7][8][9] HydF has been proposed to function as a scaffold for assembly of the H cluster 2Fe subcluster and also to mediate its subsequent insertion into HydA. [6,10] HydG has recently been shown to catalyze radical-mediated tyrosine cleavage and generate p-cresol, [11] which is a known fermentation product of several anaerobes. [12,13] The HydG-catalyzed reaction is proposed to be similar to that catalyzed by the thiamine biosynthetic enzyme ThiH and to yield dehydroglycine as an intermediate. [14][15][16] It was hypothesized by Pilet et al. [11] that two molecules of dehydroglycine condense at an FeS cluster on HydG and result in generation of the dithiolate ligand. Herein, we demonstrate that cyanide is a product of HydGcatalyzed tyrosine cleavage, a result which clarifies the role of HydG and indicates that tyrosine is the source of the cyanide ligands in the H cluster.To investigate the reaction products formed by HydG, enzyme activity assays containing chemically reconstituted HydG (on average 5.1 AE 0.5 Fe per HydG), tyrosine, AdoMet, and sodium dithionite were prepared. At selected time points, assays were stopped by acidification, and the precipitated protein was removed by centrifugation. HPLC-based analysis methods were then used to measure the concentration of reaction products in the supernatant, thus confirming the turnover of AdoMet to yield deoxyadenosine (DOA), whilst tyrosine was cleaved to yield p-cresol. [11] Cyanide was detected and quantified after derivatization by a modification of the method of Tracqui et al. [17] in which the cyanide anion reacts with naphthalene-2,3-dicarbaldehyde (NDA) 5 and a primary amine (either taurine (6) or N 1 ,N 1 -dimethylethane-1,2-diamine (7)), thus generating the fluorescent 1-cyanobenz[f]isoindole (CBI) derivatives 8 and 9 (Figure 1 A). After derivatization, HPLC analysis showed a fluorescent peak which coeluted with a standard of the CBI derivative (Figure 1 B). The components required for cyanide-forming activity were tyrosine, AdoMet, a reducing agent, and HydG. Omission of any of these components resulted in loss of activity, consi...