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.

Pyrroloquinoline quinone (PQQ) is an important redox active quinocofactor produced by a wide variety of bacteria. A key step in PQQ biosynthesis is a carbon-carbon cross-link reaction between glutamate and tyrosine side chains within the ribosomally synthesized peptide substrate PqqA. This reaction is catalyzed by the radical SAM enzyme PqqE. Previous X-ray crystallographic and spectroscopic studies suggested that PqqE, like the other members of the SPASM domain family, contains two auxiliary Fe-S clusters (AuxI and AuxII) in addition to the radical SAM [4Fe-4S] cluster. However, a clear assignment of the electron paramagnetic resonance (EPR) signal of each Fe-S cluster was hindered by the isolation of a His₆-tagged PqqE variant with an altered AuxI cluster. In this work, we are able to isolate soluble PqqE variants by using a less disruptive strep-tactin chromatographic approach. We have unambiguously identified the EPR signatures for four forms of Fe-S clusters present in PqqE through the use of multifrequency EPR spectroscopy: the RS [4Fe-4S] cluster, the AuxII [4Fe-4S] cluster, and two different clusters ([4Fe-4S] and [2Fe-2S]) bound in the AuxI site. The RS [4Fe-4S] cluster, the AuxII [4Fe-4S] cluster, and the [2Fe-2S] cluster form in the AuxI site can all be reduced by sodium dithionite, with g tensors of their reduced form determined as [2.040, 1.927, 1.897], [2.059, 1.940, 1.903], and [2.004, 1.958, 1.904], respectively. The AuxI [4Fe-4S] cluster that is determined on the basis of its relaxation profile can be reduced only by using low-potential reductants such as Ti(III) citrate or Eu(II)-DTPA to give rise to a g₁ = 2.104 signal. Identification of the EPR signature for each cluster paves the way for further investigations of SPASM domain radical SAM enzymes.

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.

Owing to their importance, diversity and abundance of generated paramagnetic species, radical S-adenosylmethionine (rSAM) enzymes have become popular targets for electron paramagnetic resonance (EPR) spectroscopic studies. In contrast to prototypic single-domain and thus single-[4Fe-4S]-containing rSAM enzymes, there is a large subfamily of rSAM enzymes with multiple domains and one or two additional iron-sulfur cluster(s) called the SPASM/twitch domain-containing rSAM enzymes. EPR spectroscopy is a powerful tool that allows for the observation of the iron-sulfur clusters as well as potentially trappable paramagnetic reaction intermediates. Here, we review continuous-wave and pulse EPR spectroscopic studies of SPASM/twitch domain-containing rSAM enzymes. Among these enzymes, we will review in greater depth four well-studied enzymes, BtrN, MoaA, PqqE, and SuiB. Towards establishing a functional consensus of the additional architecture in these enzymes, we describe the commonalities between these enzymes as observed by EPR spectroscopy.

A set of birnessite-type manganese oxides with both monovalent alkaline cations (Li+, Na+, K+, Rb+ or Cs+) and divalent alkaline- earth cations (Ca2+ or Mg2+) has been synthesized using electrolytic deposition methods. X-ray diffraction gives typical birnessite-type patterns, with diffraction peaks at 2θ = ∼12.01° and 24.7° assigned to (001) and (002) planes. Electron paramagnetic resonance spectra suggest differences in the stability of Mn(III) species in manganese-oxide layer, which is more stable in birnessite samples with divalent cations than samples with monovalent cations. The activity of samples for an anodic O2-evolution reaction is considerably affected by electrostatic potential and Lewis acidity of the monovalent cation, following the sequence of Li+ > Na+ > K+ > Rb+ > Cs+. While for samples with intercalating divalent alkaline-earth cations, there was slightly lower activity for O2 evolution, which may be correlated to different stability of Mn(III) species in the birnessite layers. In addition, we observed much higher O2-evolution activity at an overpotential of ca. 220 - 230 mV if the samples were heat-treated between 300 - 500 °C once they were electrolytically deposited as LiMnOx films. Clearly dry heating causes changes in both the crystalline structure and electronic structure of manganese-oxide layers, as indicated by associated changes in water-oxidation activity.

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.

Biotin (vitamin B₇) is an enzyme cofactor required by organisms from all branches of life but synthesized only in microbes and plants. In the final step of biotin biosynthesis, a radical S-adenosyl-l-methionine (SAM) enzyme, biotin synthase (BioB), converts the substrate dethiobiotin to biotin through the stepwise formation of two C-S bonds. Previous electron paramagnetic resonance (EPR) spectroscopic studies identified a semistable intermediate in the formation of the first C-S bond as 9-mercaptodethiobiotin linked to a paramagnetic [2Fe-2S] cluster through one of its bridging sulfides. Herein, we report orientation-selected pulse EPR spectroscopic results that reveal hyperfine interactions between the [2Fe-2S] cluster and a number of magnetic nuclei (e.g., ⁵⁷Fe, ¹⁵N, ¹³C, and ²H) introduced in a site-specific manner via biosynthetic methods. Combining these results with quantum chemical modeling gives a structural model of the intermediate showing that C6, the target of the second hydrogen-atom abstraction, is now in close proximity to the nascent thioether sulfur and is ideally positioned for the second C-S bond forming event.

The biosynthesis of the active site of the [FeFe]-hydrogenases (HydA1), the H-cluster, is of interest because these enzymes are highly efficient catalysts for the oxidation and production of H₂. The biosynthesis of the [2Fe]_H subcluster of the H-cluster proceeds from simple precursors, which are processed by three maturases: HydG, HydE, and HydF. Previous studies established that HydG produces an Fe(CO)₂(CN) adduct of cysteine, which is the substrate for HydE. In this work, we show that by using the synthetic cluster [Fe₂(μ-SH)₂(CN)₂(CO)₄]^2- active HydA1 can be biosynthesized without maturases HydG and HydE.