The active site of the [FeFe] hydrogenase (HydA1), the H-cluster, is a 6-Fe cofactor that contains CO and CN^- ligands. It undergoes several different oxidation and protonation state changes in its catalytic cycle to metabolize H₂. Among them, the well-known H_ox state and the recently identified H_hyd state are thought to be directly involved in H₂ activation and evolution, and they are both EPR active with net spin S = 1/2. Herein, we report the pulse electronic paramagnetic spectroscopic investigation of these two catalytic states in Chlamydomonas reinhardtii HydA1 ( CrHydA1). Using an in vitro biosynthetic maturation approach, we site-specifically installed ¹³C into the CO or CN^- ligands and ⁵⁷Fe into the [2Fe]_H subcluster of the H-cluster in order to measure the hyperfine couplings to these magnetic nuclei. For H_ox, we measured ¹³C hyperfine couplings (¹³CO a_iso of 25.5, 5.8, and 4.5 MHz) corresponding to all three CO ligands in the H-cluster. We also observed two ⁵⁷Fe hyperfine couplings (⁵⁷Fe a_iso of ∼17 and 5.7 MHz) arising from the two Fe atoms in the [2Fe]_H subcluster. For H_hyd, we only observed two distinct ¹³CO hyperfine interactions (¹³CO a_iso of 0.16 and 0.08 MHz) and only one for ¹³CN^- (¹³CN a_iso of 0.16 MHz); the couplings to the ¹³CO/¹³CN^- on the distal Fe of [2Fe]_H may be too small to detect. We also observed a very small (<0.3 MHz) ⁵⁷Fe HFI from the labeled [2Fe]_H subcluster and four ⁵⁷Fe HFI from the labeled [4Fe-4S]_H subcluster (⁵⁷Fe a_iso of 7.2, 16.6, 28.2, and 35.3 MHz). These hyperfine coupling constants are consistent with the previously proposed electronic structure of the H-cluster at both H_ox and H_hyd states and provide a basis for more detailed analysis.

The active site of [FeFe] hydrogenase, the H-cluster, consists of a canonical [4Fe-4S]_H subcluster linked to a unique binuclear [2Fe]_H subcluster containing three CO, two CN^- and a bridging azadithiolate (adt, NH(CH₂S^-)₂) ligand. While it is known that all five diatomic ligands are derived from tyrosine, there has been little knowledge as to the formation and installation of the adt ligand. Here, by using a combination of a cell-free in vitro maturation approach with pulse electronic paramagnetic resonance spectroscopy, we discover that serine donates the nitrogen atom and the CH₂ group to the assembly of the adt ligand. More specifically, both CH₂ groups in adt are sourced from the C3 methylene of serine.

[FeFe] hydrogenases carry out the redox interconversion of protons and molecular hydrogen (2H⁺ + 2e^- ⇌ H₂) at a complex Fe-S active site known as the H-cluster. The H-cluster consists of a [4Fe-4S] subcluster, denoted here as [4Fe]_H, linked via a cysteine sulfur to an interesting organometallic [2Fe]_H subcluster thought to be the subsite where the catalysis occurs. This [2Fe]_H subcluster consists of two Fe atoms, linked with a bridging CO and a bridging SCH₂NHCH₂S azadithiolate (adt), with additional terminal CO and CN ligands bound to each Fe. Synthesizing such a complex organometallic unit is a fascinating problem in biochemistry, complicated by the toxic nature of both the CO and CN^- species and the relative fragility of the azadithiolate bridge. It has been known for a number of years that this complex biosynthesis is carried out by a set of three essential Fe-S proteins, HydE, HydF, and HydG. HydF is a GTPase, while HydE and HydG are both members of the large family of radical S-adenosylmethionine (rSAM) enzymes. In this perspective we describe the history of research and discovery concerning these three Fe-S "maturase" proteins and describe recent evidence for a sequential biosynthetic pathway beginning with the synthesis of a mononuclear organometallic [Fe(ii)(CO)₂CN(cysteine)] complex by the rSAM enzyme HydG and its subsequent activation by the second rSAM enzyme HydE to form a highly reactive Fe(i)(CO)₂(CN)S species. In our model a pair of these Fe(i)(CO)₂(CN)S units condense to form the [Fe(CO)₂(CN)S]₂ diamond core of the [2Fe]_H cluster, requiring only the installation of the central CH₂NHCH₂ portion of the azadithiolate bridge, whose atoms are all sourced from the amino acid serine. This final step likely occurs with an interplay of HydE and HydF, the details of which yet remain to be elucidated.

Nature uses multinuclear metal clusters to catalyse a number of important multielectron redox reactions. Examples that employ complex Fe-S clusters in catalysis include the Fe-Mo cofactor (FeMoco) of nitrogenase and its V and all-Fe variants, and the [FeFe] and [NiFe] hydrogenases. This Perspective begins with a focus on the catalytic H-cluster of [FeFe] hydrogenase, which is highly active in producing molecular H₂. There has been much recent progress in characterizing the enzyme-catalysed assembly of the H-cluster, including information gleaned from spectroscopy combined with in vitro isotopic labelling of this cluster using chemical synthesis. We then compare the lessons learned from H-cluster biosynthesis to what is known about the bioassembly of the binuclear active site of [NiFe] hydrogenase and the nitrogenase active site cluster FeMoco.

The [FeFe] hydrogenase catalyzes the redox interconversion of protons and H₂ with a Fe-S "H-cluster" employing CO, CN, and azadithiolate ligands to two Fe centers. The biosynthesis of the H-cluster is a highly interesting reaction carried out by a set of Fe-S maturase enzymes called HydE, HydF, and HydG. HydG, a member of the radical S-adenosylmethionine (rSAM) family, converts tyrosine, cysteine, and Fe(II) into an organometallic Fe(II)(CO)₂(CN)cysteine "synthon", which serves as the substrate for HydE. Although key aspects of the HydG mechanism have been experimentally determined via isotope-sensitive spectroscopic methods, other important mechanistic questions have eluded experimental determination. Here, we use computational quantum chemistry to refine the mechanism of the HydG catalytic reaction. We utilize quantum mechanics/molecular mechanics simulations to investigate the reactions at the canonical Fe-S cluster, where a radical cleavage of the tyrosine substrate takes place and proceeds through a relay of radical intermediates to form HCN and a COO^•- radical anion. We then carry out a broken-symmetry density functional theory study of the reactions at the unusual five-iron auxiliary Fe-S cluster, where two equivalents of CN^- and COOH^• coordinate to the fifth "dangler iron" in a series of substitution and redox reactions that yield the synthon as the final product for further processing by HydE.

Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)₂(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN^- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster.

The radical SAM enzyme HydG generates CO- and CN^--containing Fe complexes that are involved in the bioassembly of the [FeFe] hydrogenase active cofactor, the H-cluster. HydG contains a unique 5Fe-4S cluster in which the fifth "dangler" Fe and the coordinating cysteine molecule have both been shown to be essential for its function. Here, we demonstrate that this dangler Fe can be replaced with Ni²⁺ or Co²⁺ and that the cysteine can be replaced with selenocysteine. The resulting HydG variants were characterized by electron paramagnetic resonance and X-ray absorption spectroscopy, as well as subjected to a Tyr cleavage assay. Both Ni²⁺ and Co²⁺ are shown to be exchange-coupled to the 4Fe-4S cluster, and selenocysteine substitution does not alter the electronic structure significantly. XAS data provide details of the coordination environments near the Ni, Co, and Se atoms and support a close interaction of the dangler metal with the FeS cluster via an asymmetric SeCys bridge. Finally, while we were unable to observe the formation of novel organometallic species for the Ni²⁺ and Co²⁺ variants, the selenocysteine variant retains the activity of wild type HydG in forming [Fe(CO)_x(CN)_y] species. Our results provide more insights into the unique auxiliary cluster in HydG and expand the scope of artificially generated Fe-S clusters with heteroatoms.

Radical S-adenosylmethionine (radical SAM or rSAM) enzymes use their S-adenosylmethionine cofactor bound to a unique Fe of a [4Fe-4S] cluster to generate the "hot" 5'-deoxyadenosyl radical, which drives highly selective radical reactions via specific interactions with a given rSAM enzyme's substrate. This Perspective focuses on the two rSAM enzymes involved in the biosynthesis of the organometallic H-cluster of [FeFe] hydrogenases. We present here a detailed sequential model initiated by HydG, which lyses a tyrosine substrate via a 5'-deoxyadenosyl H atom abstraction from those amino acid's amino group, initially producing dehydroglycine and an oxidobenzyl radical. In this model, two successive radical cascade reactions lead ultimately to the formation of HydG's product, a mononuclear Fe organometallic complex: [Fe(II)(CN)(CO)₂(cysteinate)]^-, with the iron originating from a unique "dangler" Fe coordinated by a cysteine ligand providing a sulfur bridge to another [4Fe-4S] auxiliary cluster in the enzyme. In turn, in this model, [Fe(II)(CN)(CO)₂(cysteinate)]^- is the substrate for HydE, the second rSAM enzyme in the biosynthetic pathway, which activates this mononuclear organometallic unit for dimerization, forming a [Fe₂S₂(CO)₄(CN)₂] precursor to the [2Fe] _H component of the H-cluster, requiring only the completion of the bridging azadithiolate (SCH₂NHCH₂S) ligand. This model is built upon a foundation of data that incorporates cell-free synthesis, isotope sensitive spectroscopies, and the selective use of synthetic complexes substituting for intermediates in the enzymatic "assembly line". We discuss controversies pertaining to this model and some remaining open issues to be addressed by future work.

The enzyme [FeFe]-hydrogenase (HydA1) contains a unique 6-iron cofactor, the H-cluster, that has unusual ligands to an Fe-Fe binuclear subcluster: CN^-, CO, and an azadithiolate (adt) ligand that provides 2 S bridges between the 2 Fe atoms. In cells, the H-cluster is assembled by a collection of 3 maturases: HydE and HydF, whose roles aren't fully understood, and HydG, which has been shown to construct a [Fe(Cys)(CO)₂(CN)] organometallic precursor to the binuclear cluster. Here, we report the in vitro assembly of the H-cluster in the absence of HydG, which is functionally replaced by adding a synthetic [Fe(Cys)(CO)₂(CN)] carrier in the maturation reaction. The synthetic carrier and the HydG-generated analog exhibit similar infrared spectra. The carrier allows HydG-free maturation to HydA1, whose activity matches that of the native enzyme. Maturation with ¹³CN-containing carrier affords ¹³CN-labeled enzyme as verified by electron paramagnetic resonance (EPR)/electron nuclear double-resonance spectra. This synthetic surrogate approach complements existing biochemical strategies and greatly facilitates the understanding of pathways involved in the assembly of the H-cluster. As an immediate demonstration, we clarify that Cys is not the source of the carbon and nitrogen atoms in the adt ligand using pulse EPR to target the magnetic couplings introduced via a ¹³C₃,¹⁵N-Cys-labeled synthetic carrier. Parallel mass-spectrometry experiments show that the Cys backbone is converted to pyruvate, consistent with a cysteine role in donating S in forming the adt bridge. This mechanistic scenario is confirmed via maturation with a seleno-Cys carrier to form HydA1-Se, where the incorporation of Se was characterized by extended X-ray absorption fine structure spectroscopy.