Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

Abstract

H 2 metabolism in microorganisms requires the catalytic activity of hydrogenases, a specialized group of metalloproteins (29,38,51 (8,10,12,28,37,42,45). In contrast, characterization of [FeFe] hydrogenase maturation has been limited by a lack of knowledge of the proteins functioning in the biosynthesis of the catalytic H cluster. Recently, we identified two genes in Chlamydomonas reinhardtii, hydEF and hydG, which encode proteins required for [FeFe] hydrogenase activity by functioning in H cluster biosynthesis and enzyme maturation (40). Both proteins contain conserved motifs found in radical S-adenosylmethionine (SAM)-binding proteins, suggesting that biosynthesis of the H cluster during [FeFe] hydrogenase maturation involves radical-SAM-dependent mechanisms. The radical-SAM domains in both HydE and HydG contain the conserved motif Cx 3 Cx 2 C, with additional motifs in the C-terminal ends that are characteristic of iron-sulfur cluster-binding sites (16). The HydF protein differs from HydE and HydG in that it belongs to the GTPase protein family (27). At the N-terminal end of HydF, there are motifs that match the conserved GTP-binding motifs, with a potential iron-sulfur cluster-binding motif present at the C-terminal end (27).Although the biochemistry of [FeFe] hydrogenase maturation is an emerging field of investigation, the [FeFe] hydrogenases from several organisms have been extensively investigated by biochemical, spectroscopic, and structural methods (reviewed in reference 1). As a result of these investigations, a number of the characteristics of the H cluster and catalytic site have been elucidated. The H cluster is arranged as a unique [2Fe] center connected by a conserved cysteine to a [4Fe4S] cubane cluster (1,35,38). The iron atoms of the [2Fe] center are bridged by a dithio moiety which, based on a combination of structural and theoretical studies, is interpreted as either a propanedithiol or a di(thiomethyl)amine (13,34,35,38). Other features of the [2Fe] center are asymmetric CN and CO ligand pairs at each iron, with an additional CO bridging the two iron atoms (35,38,39). Thus, the HydEF and HydG proteins are thought to function together in a series of undefined steps in synthesis and insertion of the chemically complex H cluster at the [FeFe] hydrogenase catalytic site during maturation.As part of our previous report, we demonstrated that anaerobic coexpression of the C. reinhardtii [FeFe] hydrogenase maturation and structural genes in Escherichia coli resulted in the biosynthesis of an active [FeFe] hydrogenase (40). However, the C. reinhardtii hydEF and hydG clones were unstable and susceptible to rearrangements in E. coli, a problem that

HydF as a scaffold protein in [FeFe] hydrogenase H‐cluster biosynthesis

“…HydA from Clostridium pasteurianum was cloned as described previously [5] modified for the presence of an N-terminal 6x-histidine tag. Constructs were confirmed by DNA sequencing.…”

“…Recent studies involving the analysis of mutants of Chlamydomonas reinhardtii defective in hydrogen production have revealed that products of the hydEF and hydG genes are required for the accumulation of active [FeFe] hydrogenase, and that hydE, hydF, and hydG are common to all organisms in which active [FeFe] hydrogenases are found [4]. Further studies have shown that the coexpression in Escherichia coli of HydE, HydF, and HydG from Clostridium acetobutylicum with the HydA [FeFe] hydrogenase protein from a variety of microbial sources enables the formation of active [FeFe] hydrogenases [5]. Genome annotation indicates that HydE and HydG are members of the radical Sadenosylmethionine (AdoMet) enzyme family, and that HydF is likely to be a GTPase [4].…”

Hydrogen production at high Faradaic efficiency by a bio-electrode based on TiO2 adsorption of a new [FeFe]-hydrogenase from Clostridium perfringens

“…Recombinant expression of the hydrogenase CpHydA from Clostridium perfringens SM09 [22] was performed in E. coli 30 Rosetta2(DE3) as previously described [31] upon transformation of the competent cells with plasmid pECPF2655, obtained by ligating the entire coding sequence of CPF_2655 into the empty expression vector pECr1 [23,24] between NdeI and XhoI sites. After the induction, cells were incubated over night under pure argon flow to maintain anaerobic conditions in a water bath at 30°C.…”

“…In this work, a novel [FeFe]-hydrogenase CpHydA (encoded by CPF_2655 gene) from a Clostridium perfringens strain newly isolated in a pilot plant with high hydrogen productivity [21][22][23] was immobilized by adsorption on stationary TiO2 electrodes. The heterologous expression in E. coli was achieved thanks to a previously established system [23,24] and it allowed to obtain a pure and active protein in good yields. Anatase TiO2 was used because of its ability to bind hydrogenases [9] with a favourable orientation for electron transfer by simple adsorption; this is possibly due to a major The resulting hydrogen producing bioelectrode was validated by chronoamperometric experiments and tested for production of hydrogen gas by quantitative measurement in gas chromatography.…”

Crucial Role of Conserved Cysteine Residues in the Assembly of Two Iron−Sulfur Clusters on the CIA Protein Nar1

“…This moiety is comprised of the proximal [4Fe-4S] cluster and a connected dithiolate-, CO-, and CN-containing 2Fe center which serves as the active site (21). In bacteria, trichomonads, and possibly algae, synthesis of the H-cluster depends on the specialized biogenesis factors (termed HydEFG) which are not conserved in other eukaryotes (25)(26)(27)(28). Hence, it seems unlikely that eukaryotic Nar1 proteins contain an H-type catalytic center, rendering the function of the C-terminal cysteine residues unclear.…”