Serine is the molecular source of the NH(CH2)2 bridgehead moiety of the in vitro assembled [FeFe] hydrogenase H-cluster

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.

The CH₂NHCH₂ bridgehead moiety of the FeFe] hydrogenase H-cluster is derived from serine as revealed by isotope labeling and EPR spectroscopy.

Hydrogenases catalyze the reversible reactions of H₂ oxidation and proton reduction, and are involved in many microbial metabolic pathways.¹ FeFe] hydrogenases in particular are hyper-efficient, with turnover rates up to 10⁴/s.² This has led to intense focus on FeFe] hydrogenases for sustainable production of H₂ and the design of fuel cells.³ The active site of FeFe] hydrogenases is a six-iron cofactor called the H-cluster (Scheme 1), which consists of a canonical cuboid 4Fe–4S]_H subcluster linked through a bridging cysteine (Cys) residue to a binuclear 2Fe]_H subcluster in which the two iron ions are coordinated by three CO, two CN⁻ and an azadithiolate (adt, NH(CH₂S⁻)₂) bridging ligand. The 2Fe]_H subcluster has been proposed to be the site for H₂ binding and hydride formation,^4,5 which serves as a natural blueprint for designing small molecule catalysts for hydrogen evolution reactions.⁶ The unique structure and catalytic activity has thus raised much interest in the biosynthesis of the H-cluster, which poses a great challenge in cofactor assembly that involves toxic ligands, oxygen sensitivity and an organic adt ligand that has little inherent stability.Open in a separate windowScheme 1Bioassembly of the H-cluster highlighting the source of each moiety.While the 4Fe–4S]_H subcluster in the H-cluster can be formed by the housekeeping gene products that are used to assemble such standard Fe–S clusters, the in vivo bioassembly of the unique 2Fe]_H subcluster requires three special Fe–S “maturase” proteins: HydE, HydF, and HydG.^7,8 Although the functions of HydE and HydF have not been fully elucidated,^9–12 recent studies indicate that HydG is a bifunctional 4Fe–4S radical S-adenosyl-l-methionine (SAM) enzyme which lyses tyrosine to generate CO and CN⁻ and forms a (Cys)Fe(CO)₂(CN)] organometallic precursor to the H-cluster on a dangler Fe(Cys) site in HydG.^13–16 More recently, by using a synthetic (Cys)Fe(CO)₂(CN)] carrier we have shown that the two sulfur atoms in the adt ligand are derived from the precursor-bound Cys, but that the CH₂NHCH₂ component is not.¹⁷ Taken together, the biosynthetic origins of the Fe₂S₂(CO)₃(CN)₂] part of the 2Fe]_H subcluster are depicted in Scheme 1: all five diatomic ligands are tailored from tyrosine by HydG;¹⁸ the two sulfur atoms and the two Fe atoms are from the dangler Fe(Cys) site in HydG (which can be reconstituted with Fe²⁺ and free Cys in solution¹⁹). Remarkably, these components are all delivered to the binuclear cluster assembly in the form of the (Cys)Fe(CO)₂(CN)] product of HydG. Given these recent advances, the only missing part of the puzzle is the crucial NH(CH₂)₂ moiety: what are its molecular precursors? It has been hypothesized that HydE, which is also a 4Fe–4S radical SAM enzyme, may be involved in the formation of adt, though its physiological substrate and reaction mechanism remains under investigation.^9,10 As for any enzymatic reaction, knowing the actual substrate(s) for the reaction is crucial for unraveling the ultimate mechanism. Therefore, determining of molecular sourcing of the CH₂NHCH₂ component of the adt bridge, currently unknown, is the focus of this work.Assembly of the H-cluster in the lab can be achieved by semi-synthetic and biochemical approaches other than directly co-expressing hydA, hydE, hydF and hydG genes in cells. One very useful method alleviates the need for HydG, HydE, and in some cases, HydF, by using a synthetic Fe₂(adt)(CO)₄(CN)₂] complex as a direct donor to the 2Fe]_H subcluster assembly.^20–22 Another “cell free synthesis” approach uses HydE/F/G in an in vitro H-cluster maturation reaction developed by the Swartz group.^23,24 The specific in vitro maturation reaction used in our current investigation contains a mixture of E. coli cell lysate containing separately overexpressed HydE, HydF, HydG (all from Shewanella oneidensis), apo-HydA1 (from Chlamydomonas reinhardtii) that harbors the 4Fe-4S]_H subcluster, and a cocktail of low molecular weight cofactors and precursors.²³ This biochemical approach gives us the opportunity to use the same set of enzymes that build the H-cluster in cells, but also enables us to determine the molecular source of each of the components in the H-cluster by using isotope-labeled cofactors/precursors, a procedure that would be very difficult to carry out and fully control in vivo. For example, by supplementing 1-¹³C-Tyr or 2-¹³C-Tyr into the in vitro maturation reaction, the CO or CN⁻ ligands to the diiron subcluster of the maturated HydA1 are respectively labeled with ¹³C.^25,26 The presence of these ¹³C labels can then in turn be detected and analyzed by using advanced electron paramagnetic resonance (EPR) spectroscopy to measure the hyperfine couplings between the magnetic ¹³C nuclei and the unpaired electron spin distributed over the H-cluster in its redox-poised paramagnetic states. In this work, we now search for the source(s) of the CH₂NHCH₂ moiety by using a similar strategy of in vitro maturation coupled to high resolution EPR to screen the assembly products formed with various isotopically labeled small molecule candidates. The presence of nitrogen element in the CH₂NHCH₂ fragment suggests an amino acid origin as one possibility. A systematic screening by pulse EPR of the in vitro maturation products generated with ¹³C, ¹⁵N, and ²H-labeled amino acids reveals that serine (Ser) serves as a molecular source for the NH(CH₂)₂ moiety of the H-cluster.