Biosynthesis of the unusual organometallic H-cluster at the active site of the [FeFe]-hydrogenase requires three accessory proteins, two of which are radical AdoMet enzymes (HydE, HydG) and one of which is a GTPase (HydF). We demonstrate here that HydG catalyzes the synthesis of CO using tyrosine as a substrate. CO production was detected by using deoxyhemoglobin as a reporter and monitoring the appearance of the characteristic visible spectroscopic features of carboxyhemoglobin. Assays utilizing (13)C-tyrosine were analyzed by FTIR to confirm the production of HbCO and to demonstrate that the CO product was synthesized from tyrosine. CO ligation is a common feature at the active sites of the [FeFe], [NiFe], and [Fe]-only hydrogenases; however, this is the first report of the enzymatic synthesis of CO in hydrogenase maturation.

The [FeFe]-hydrogenase catalytic site H cluster is a complex iron sulfur cofactor that is sensitive to oxygen (O2). The O2 sensitivity is a significant barrier for production of hydrogen as an energy source in water-splitting, oxygenic systems. Oxygen reacts directly with the H cluster, which results in rapid enzyme inactivation and eventual degradation. To investigate the progression of O2-dependent [FeFe]-hydrogenase inactivation and the process of H cluster degradation, the highly O2-sensitive [FeFe]-hydrogenase HydA1 from the green algae Chlamydomonas reinhardtii was exposed to defined concentrations of O2 while monitoring the loss of activity and accompanying changes in H cluster spectroscopic properties. The results indicate that H cluster degradation proceeds through a series of reactions, the extent of which depend on the initial enzyme reduction/oxidation state. The degradation process begins with O2 interacting and reacting with the 2Fe subcluster, leading to degradation of the 2Fe subcluster and leaving an inactive [4Fe-4S] subcluster state. This final inactive degradation product could be reactivated in vitro by incubation with 2Fe subcluster maturation machinery, specifically HydF(EG), which was observed by recovery of enzyme activity.

a b s t r a c tIron-sulfur cluster coordination was probed in the [FeFe]-hydrogenase H cluster maturation scaffold HydF. Putative Cys thiol and His imidazole ligation identified through multiple sequence alignments and structural studies were subjected to amino acid substitution and the variants were biochemically characterized. The results implicate a role for C304, C353, C356, and H306 of Clostridium acetobutylicum HydF in FeS cluster binding. Individual ligand substitutions affect both [4Fe-4S] and [2Fe-2S] cluster coordination suggesting shared coordination or cluster interconversion. Substitutions at C353 and H306 appear to preferentially impact the presence of the [2Fe-2S] cluster complement of the resulting variants of HydF. The results implicate a potential role for these residues in biosynthesis specifically and potential in bridging the [4Fe-4S] cluster to 2Fe subcluster biosynthetic intermediates. Structured summary of protein interactions:HydF and HydF bind by molecular sieving(View interaction: 1, 2)

The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H2 or other reduced products has been limited by their sensitivity to oxygen (O2). Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O2 gradients in a vertical water column of Great Salt Lake (GSL), UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O2 concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H2 transfer and O2 susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O2 tolerant enzymes for biotechnological applications.

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Carbon monoxide (CO) and cyanide (CN–) can act as potent inhibitors of enzymes necessary for primary biochemical processes, however they also play important roles in biological systems. Well‐studied cases include CN– biosynthesis in plants to act as a defense against herbivores and pathogens, CN– biosynthesis in certain species of bacteria to remove excess glycine, and CO biosynthesis by microbes for energy metabolism in the Wood–Ljungdahl pathway. The utilization of CO and CN– as essential metal ligands in biology is even more limited, with the only known examples being at the active sites of hydrogenase enzymes. This class of enzymes catalyzes the reversible oxidation of hydrogen, a reaction that in biology appears to be entirely dependent on the presence of CO and/or CN– ligands. To date, synthetic mimicsof hydrogenase active sites have not reproduced hydrogen production rates observed in some hydrogenases; it is thus of considerable interest to understand how biology has solved the intriguing problem of biosynthesizing efficient hydrogen catalysts. Of the hydrogenase enzymes discussed herein, recent advances in the [FeFe]‐hydrogenase family has provided important insights into the synthesis of the CO and CN– ligands for its active site (H‐cluster). Biosynthesis of the complex [FeFe]‐hydrogenase active site requires only three iron–sulfur cluster‐containing maturation proteins, where two act as radical S‐adenosylmethionine (AdoMet) enzymes (HydE and HydG) and the other as a GTPase (HydF). In this review, biological CO and CN– genesis mechanisms will be assessed with specific focus on [FeFe]‐hydrogenase maturation.

This article highlights recent advances in our understanding of the biosynthetic pathway for active site H‐cluster assembly in [ FeFe ]‐hydrogenases. The H‐cluster is composed of a [ 4Fe–4S ] cubane bridged via one cysteine thiolate to a 2Fe subcluster; the 2Fe subcluster is further coordinated by carbon monoxide, cyanide, and bridging dithiolate ligands. Biosynthesis of the H‐cluster occurs through stepwise modification of simple Fe–S cluster precursors; these chemical modifications are carried out by three gene products denoted HydE , HydF , and HydG . Maturation of the [ FeFe ]‐hydrogenase requires the presence of a preformed [ 4Fe–4S ] cluster, indicating that HydE , HydF , and HydG are directed toward the biosynthesis of the 2Fe subcluster. HydE and HydG are both radical S ‐adenosylmethionine ( SAM ) enzymes and utilize a CX 3 CX 2 C motif to bind site‐differentiated [ 4Fe–4S ] 2+/+ clusters that promote the reductive cleavage of SAM into methionine and a 5′‐deoxyadenosyl radical responsible for hydrogen atom abstraction from substrate. In an extraordinary biochemical reaction, HydG uses this chemistry to catalyze the radical degradation of tyrosine into p ‐cresol and a glycine‐like intermediate; an accessory, C‐terminal [ 4Fe–4S ] cluster then degrades the latter intermediate into cyanide and carbon monoxide. HydE is presumed to be responsible for bridging dithiolate ligand biosynthesis from an unknown substrate. HydF contains an N‐terminal GTPase domain and a C‐terminal Fe–S ‐cluster‐binding domain and binds both [ 2Fe–2S ] and [ 4Fe–4S ] clusters; evidence suggests that the former cluster is modified by HydE and HydG to form a 2Fe H‐cluster precursor on HydF . The role of GTP hydrolysis in H‐cluster maturation remains largely unexplained; however, it may be related to gating the protein–protein interactions between HydF and HydE and HydG . The reactions catalyzed by HydE and HydG can be thought of as specific ligand modification events designed to fine‐tune H‐cluster reactivity; not only do the chemical transformations themselves have direct links to reactions important on early Earth, but radical SAM enzymes with their canonical CX 3 CX 2 C motif are likely very ancient. The modern diversity that is observed in chemical reactions catalyzed by the radical SAM superfamily can be explained evolutionarily through the recruitment of distinct protein domains that impart specific substrate activation capabilities. Recruitment of these domains is discussed in the context of HydE and HydG and important links are made to the nitrogenase system where intriguing parallels between [ FeFe ]‐hydA H‐cluster biosynthesis and nitrogenase FeMo ‐cofactor assembly clearly exist.

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