title: Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA(DeltaEFG).
abstract: 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 metabolism. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases (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 ligands. 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 unclear. Here we show the structure of HydA(DeltaEFG) (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 machinery and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery. 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.
authors.affiliation: Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, Montana 59717, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Mulder
authors.forename: Eric S
authors.initials: ES
authors.lastname: Boyd
authors.forename: Ranjana
authors.initials: R
authors.lastname: Sarma
authors.forename: Rachel K
authors.initials: RK
authors.lastname: Lange
authors.forename: James A
authors.initials: JA
authors.lastname: Endrizzi
authors.forename: Joan B
authors.initials: JB
authors.lastname: Broderick
authors.forename: John W
authors.initials: JW
authors.lastname: Peters
mesh_terms: D020134:Catalytic Domain; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D003013:Clostridium / Q000201:enzymology; D018360:Crystallography, X-Ray; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008958:Models, Molecular; D009591:Nitrogenase / Q000378:metabolism; D010802:Phylogeny; D011487:Protein Conformation; D013455:Sulfur / Q000378:metabolism
chemical_list: D013455:Sulfur; D006859:Hydrogen; D007501:Iron; D006864:Hydrogenase; D009591:Nitrogenase