H-Cluster assembly during maturation of the [FeFe]-hydrogenase

Abstract

The organometallic H-cluster at the active site of the [FeFe]-hydrogenase serves as the site of reversible binding and reduction of protons to produce H2. The H-cluster is unique in biology, and consists of a 2Fe subcluster tethered to a typical [4Fe-4S] cluster by a single cysteine ligand. The remaining ligands to the 2Fe subcluster include three carbon monoxides, two cyanides, and a dithiomethylamine. This mini-review will focus on the significant advances in recent years in understanding the pathway for H-cluster biosynthesis, as well as the structures, roles, and mechanisms of the three enzymes directly involved.

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

“…1A) (2,3). The biosynthesis of the H cluster has garnered much attention (4,5) given its unusual structure and exceptional H 2 production activity (6). 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).…”

“…The biosynthesis of the H cluster has garnered much attention (4,5) given its unusual structure and exceptional H 2 production activity (6). 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).…”

“…Significant progress has been made toward elucidating the individual functions of the maturases (5). HydG is a member of the radical S-adenosyl-L-methionine (SAM) family of enzymes (16) and performs a complex reaction in which Tyr and Fe are transformed into an [Fe(CO) 2 (CN)] synthon that is eventually incorporated into the [2Fe] H subcluster (17) (Fig.…”

Metallocofactor assembly for [FeFe]-hydrogenases

“…However, despite all of this hard won understanding, the natural substrate and products for HydE remain elusive. One putative role for HydE is catalyzing the biosynthesis of the azadithiolate ligand [34,35], but measuring the formation of this ligand is experimentally challenging, as in free aqueous solution such geminal hemithioaminal groups are inherently unstable and undergo hydrolysis (to formaldehyde, sulfide and ammonia in this case). Following the metabolic fate of 15 N labelled precursors through in vitro maturation may circumvent this problem [36].…”

“…Models for cluster assembly by HydF agree that the [4Fe-4S] is assembled by housekeeping iron sulfur cluster biosynthetic enzymes. Two alternatives have been considered [34] for assembly of the [2Fe] subcluster: (i) housekeeping enzymes also insert a [2Fe-2S] cluster which is modified by ligands introduced by transfer from HydG/HydE or (ii) the [2Fe] subcluster is derived from two equivalents of the Fe(CO) 2 CN synthon produced by HydG. Although many unresolved questions remain, using a biosynthetic cocktail (including maturases, substrates and cofactors) to achieve efficient transfer of labelled 57 Fe from HydG to the [2Fe] H subcluster of HydA [16] supports model (ii).…”