Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydAΔEFG

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 (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-, hydFand 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. The biosynthesis and assembly of active-site metallo-cofactors requires multiple enzymes, scaffolds and carriers. For [FeFe]hydrogenases, the gene products HydE, HydF and HydG are required for the maturation of the active-site H-cluster (Fig. 1). These gene products function to couple radical S-adenosyl-L-methionine (SAM) chemistry and nucleotide binding and hydrolysis to ligand synthesis, cluster assembly and insertion, and, ultimately, [FeFe]-hydrogenase maturation. Although several plausible schemes have been proposed for the generation of the carbon monoxide, cyanide and dithiolate ligands at the Fe site, including radical SAM-mediated sulphur insertion coupled to the decomposition or condensation of amino acids, the precise mechanism by which the various enzymes, scaffolds and carriers coordinate H-cluster maturation is unknown. Owing to their high catalytic rates of hydrogen production, much interest surrounds [FeFe]-hydrogenases as alternative biological catalysts to those containing precious metals such as platinum in hydrogen-fuelcell technology. Advancements in understanding how the H-cluster is synthesized by HydE, HydF and HydG could contribute significantly to both the genetic engineering of hydrogen-producing microorganisms and the synthesis of biomimetic hydrogen-production catalysts. HydF has been shown to transfer a cluster precursor to HydA in the final stage of [FeFe]-hydrogenase maturation and may act as a scaffold on which an H-cluster precursor is assembled. Our most recent results indicate that the H-cluster maturation machinery (the activities of HydE, HydF and HydG) is directed at the synthesis of only the 2Fe unit of the 6Fe cluster, and that the [4Fe–4S] subcluster can be synthesized independently. To better understand the synthesis and insertion of the individual [4Fe–4S]-subcluster and 2Fesubcluster components of the H-cluster during [FeFe]-hydrogenase maturation, we determined the X-ray crystal structure of HydA. We determined the structure of HydA from Chlamydomonas reinhardtii, heterologously expressed in Escherichia coli, by molecular replacement, using the structure of the [FeFe]-hydrogenase from Clostridium pasteurianum (CpI) as a search model, and refined it to a resolution of 1.97 A (Fig. 2a). We focused the study on the [FeFe]-hydrogenase from C. reinhardtii because our complementary biochemical and spectroscopic analyses examining maturation were conducted using this enzyme. In addition, C. reinhardtii HydA is of biotechnological interest and no structural information about it yet exists. The overall structure of C. reinhardtii HydA (Fig. 2a) is similar to that observed for active-site domains of the previously characterized [FeFe]-hydrogenases from C. pasteurianum (Fig. 2c) and Desulfovibrio