Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA(DeltaEFG).

Hydrogen metabolism is facilitated by the activity of three metalloenzymes: the [NiFe]- and [FeFe]-hydrogenases and the Hmd or [Fe]-hydrogenases. The catalytic core of the [FeFe]-hydrogenase (HydA) exists as a [4Fe–4S] cluster linked to a modified 2Fe subcluster. In this paper, Mulder et al. solved the X-ray crystal structure of HydA expressed in a genetic background that did not contain three other hydrogenase biosynthetic genes — the structure reveals a [4Fe–4S] cluster and an open pocket for the 2Fe subcluster. This means that H-cluster synthesis occurs in a stepwise manner with synthesis and insertion of the [4Fe–4S] subcluster occurring first, followed by synthesis of the 2Fe subcluster by specialized maturation machinery and insertion of the subcluster via a cationically charged channel that collapses following incorporation. Hydrogen metabolism is facilitated by the activity of three hydrogenase enzymes. The catalytic core of the [FeFe]-hydrogenase (HydA), called the H-cluster, exists as a [4Fe4S] subcluster linked to a modified 2Fe subcluster. Here, Chlamydomonas reinhardtii HydA was expressed in a genetic background that did not contain the other hydrogenase biosynthetic genes. The structure of this HydA was then solved, revealing the stepwise manner by which the H-cluster is synthesized, and offering insight into how HydA might have evolved. Complex enzymes containing Fe–S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism1,2. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases3,4 (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe–4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands5,6. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear7,8,9,10. Here we show the structure of HydAΔEFG (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe–4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe–4S] subcluster by generalized host-cell machinery11,12 and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery7,8,9,10. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes.