[FeFe]-Hydrogenase Maturation: Insights into the Role HydE Plays in Dithiomethylamine Biosynthesis

Abstract

HydE and HydG are radical S-adenosylmethionine enzymes required for the maturation of [FeFe]-hydrogenase (HydA) and produce the non-protein organic ligands characteristic of its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster) is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains two carbon monoxide, three cyanide, and a bridging dithiomethylamine as ligands. While recent studies have shed light on the nature of diatomic ligand biosynthesis by HydG, little information exists on the function of HydE. Herein, we present biochemical, spectroscopic, bioinformatics, and molecular modeling data that together map the active site and provide significant insight into the role of HydE in H-cluster biosynthesis. Electron paramagnetic resonance and UV-visible spectroscopic studies demonstrate that reconstituted HydE binds two [4Fe-4S] clusters and copurifies with S-adenosyl-L-methionine. Incorporation of deuterium from D2O into 5’-deoxyadenosine, the cleavage product of S-adenosyl-L-methionine, coupled with molecular docking experiments suggests that the HydE substrate contains a thiol functional group. This information, along with HydE sequence similarity and genome context networks, have allowed us to redefine the presumed mechanism for HydE away from BioB-like sulfur insertion chemistry; these data collectively suggest that the source of the sulfur atoms in the dithiomethylamine bridge of the H-cluster are likely derived from HydE’s thiol containing substrate.

Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster

“…Whereas the [4Fe-4S] H subcluster is inserted by the housekeeping Fe-S cluster machinery, the [2Fe] H subcluster is synthesized and inserted by three accessory proteins: the HydE, HydF, and HydG maturases (5,(7)(8)(9). Both L-tyrosine (Tyr) and L-cysteine (Cys) have been shown to stimulate in vitro [2Fe] H subcluster biosynthesis (10,11) with Tyr serving as the precursor to the CO and CN -ligands (12)(13)(14); the role of Cys in H-cluster maturation is less clear and an emerging area of focus (15).…”

“…1B). The substrate and product of the radical SAM enzyme HydE are presently unknown, although it is thought that HydE plays a role in building the azadithiolate ligand (5,15). HydG and HydE are thought to function in concert with the GTP-hydrolyzing enzyme HydF (18,19) to generate a [2Fe] H subcluster-like precursor (20)(21)(22) that is transferred to the hydrogenase apoprotein (apo-HydA) to yield the mature H cluster.…”

CO and CN − syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

“…Work from several laboratories has shown that the maturation of the [FeFe] center requires at least three protein maturases: HydF that has GTPase activity and appears to be both a [FeFe] center scaffold and carrier (8,9), HydG that synthesizes CO and CN − from tyrosine (10)(11)(12)(13), and HydE that, by elimination, should be involved in the synthesis of the DTMA bridge (14,15). Both HydE and HydG are members of the large radical S-adenosyl-L-methionine (SAM) protein family (16,17).…”

Metallocofactor assembly for [FeFe]-hydrogenases

“…The structure of HydE (Figure 4a) includes a large cavity (991 Å 3 ) within the TIM barrel that is well suited to bind a second substrate (in addition to SAM). Efforts to identify this substrate include in silico [24] and in vitro screening experiments [32,33]. The rate of SAM turnover to form 5ʹ-deoxyadenosine was recently used to screen a range of putative HydE second substrates, leading to the conclusion that the HydE substrate likely contains a thiol [33].…”

“…Efforts to identify this substrate include in silico [24] and in vitro screening experiments [32,33]. The rate of SAM turnover to form 5ʹ-deoxyadenosine was recently used to screen a range of putative HydE second substrates, leading to the conclusion that the HydE substrate likely contains a thiol [33]. Thiocyanate was identified as a fragment with significant affinity for HydE [30] and more recent studies with HydE crystals demonstrated that a small molecule, (2R,4R)-2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid, could intercept the 5ʹ-deoxyadenosyl radical intermediate, forming a new 5ʹ-C to sulfur bond in the product [32].…”