Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase.

To identify genes necessary for the photoproduction of H(2) in Chlamydomonas reinhardtii, random insertional mutants were screened for clones unable to produce H(2). One of the identified mutants, denoted hydEF-1, is incapable of assembling an active [Fe] hydrogenase. Although the hydEF-1 mutant transcribes both hydrogenase genes and accumulates full-length hydrogenase protein, H(2) production activity is not observed. The HydEF protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [Fe] hydrogenase. In the C. reinhardtii genome, the HydEF gene is adjacent to another hydrogenase-related gene, HydG. All organisms with [Fe] hydrogenase and sequenced genomes contain homologues of HydE, HydF, and HydG, which, prior to this study, were of unknown function. Within several prokaryotic genomes HydE, HydF, and HydG are found in putative operons with [Fe] hydrogenase structural genes. Both HydE and HydG belong to the emerging radical S-adenosylmethionine (commonly designated "Radical SAM") superfamily of proteins. We demonstrate here that HydEF and HydG function in the assembly of [Fe] hydrogenase. Northern blot analysis indicates that mRNA transcripts for both the HydEF gene and the HydG gene are anaerobically induced concomitantly with the two C. reinhardtii [Fe] hydrogenase genes, HydA1 and HydA2. Complementation of the bx;1C. reinhardtii hydEF-1 mutant with genomic DNA corresponding to a functional copy of the HydEF gene restores hydrogenase activity. Moreover, co-expression of the C. reinhardtii HydEF, HydG, and HydA1 genes in Escherichia coli results in the formation of an active HydA1 enzyme. This represents the first report on the nature of the accessory genes required for the maturation of an active [Fe] hydrogenase.

Hydrogen has enormous potential to serve as a non-polluting fuel, alleviating the environmental and political concerns asso-ciated with fossil energy utilization. Among the most efficient H 2 -generating catalysts known are the [Fe] hydrogenase enzymes, which are found in numerous microorganisms, including the photosynthetic green alga Chlamydomonas reinhardtii (1,2). Our research, aimed at identifying accessory proteins required for H 2 production in C. reinhardtii, has resulted in the discovery of two [Fe] hydrogenase assembly genes. These genes are essential for the formation of an active [Fe] hydrogenase and are conserved among sequenced organisms containing [Fe] hydrogenase enzymes.
Algal H 2 production was first reported by Hans Gaffron and co-workers in seminal experiments performed over 60 years ago (3). Photosynthetic green algae are unique in that H 2 production by [Fe] hydrogenases is coupled directly to water oxidation through photosystem II and the photosynthetic electron transport chain, providing the means to generate H 2 using sunlight. In the past 5 years, great strides have been made in sustaining H 2 photoproduction activity in C. reinhardtii (4,5), and intensive research continues to probe novel ways to use this and other photosynthetic organisms to generate renewable H 2 .
Hydrogenases containing metallo-catalytic clusters occur as [NiFe] or [Fe]-only enzymes (2,6,7). These two forms are phylogenetically distinct (8), which suggests that hydrogenase function is the result of convergent evolution (2). Although [NiFe] and [Fe] hydrogenases are genetically unrelated, similarities between the proteins do exist. First, the active sites of both enzymes contain CO and CN ligands, and, second, each active site contains a binuclear metal center. In general, [NiFe] hydrogenases catalyze H 2 uptake, whereas [Fe] hydrogenases tend to catalyze H 2 evolution (9). Furthermore, the turnover number of [Fe] hydrogenases is 10 -100 times higher than that of [NiFe] hydrogenases (9), making the former one of the most efficient H 2 production catalysts known. Hydrogenases containing NiFeSe (10) and a unique class of hydrogenases containing a single Fe atom bound to a co-factor have also been reported (11).
The [NiFe] hydrogenases are found in a variety of anaerobic and facultative archaea, eubacteria, and cyanobacteria, and they typically consist of two distinct peptides that are referred to as the large and small subunits (2,12). Structural data obtained from x-ray crystallographic studies show a catalytic site containing a Ni-Fe binuclear center buried inside the large subunit (13,14).
The [Fe] hydrogenases are found in strictly anaerobic bacteria such as Clostridium and Desulfovibrio, as well as in some green algae and several eukaryotic protists with hydrogenosomes such as Trichomonas (2). These enzymes may exist as distinct monomers or heteromers, and x-ray crystallographic structural data are available for CpI from Clostridium pasteurianum (15) and DdH from Desulfovibrio desulfuricans (16). The [Fe] hydrogenase catalytic site is known as the H-cluster and consists of a [4Fe4S] cluster connected through a bridging cysteinyl ligand to a binuclear [2Fe] center (15,16). In addition to binding CO and CN (17), the iron atoms of the [2Fe] center coordinate a bridging organic group thought to be a di(thiomethyl)amine moiety (18). The H-clusters of [Fe] hydrogenases are easily oxidized and are located in the interior of the protein structure. These sites are connected to the surface by a hydrophobic channel that facilitates H 2 diffusion. The additional [FeS] clusters required for electron transport from/to soluble mediators are also found in most [Fe] hydrogenase enzymes (2,15,16); however, algal [Fe] hydrogenases lack these additional metal clusters (19,20).
The assembly and insertion of metal clusters into metalloenzymes often require specific accessory proteins (7,21,22). The complexity of the [Fe] hydrogenase H-cluster suggests that additional proteins must be involved in the assembly of an active [Fe] hydrogenase, as is the case with the [NiFe] hydrogenases (2,7). However, until now no accessory genes involved in the biosynthesis and activation of [Fe] hydrogenase have been identified definitively, with the possible exception of the protein TM1420 in Thermotoga maritima. In this organism, an open reading frame encoding an [FeS] protein, TM1420, was identified in the hydrogenase operon. Characterization of TM1420 purified from Escherichia coli led to the suggestion that it may function in the assembly of the T. maritima [Fe] hydrogenase (23); however, its mode of action was not characterized, and TM1420 may be specific for the heterotrimeric T. maritima [Fe] hydrogenase.
Hydrogen and Oxygen Assays-Chemochromic screening was performed by using colonies growing on Tris acetate-phosphate agar plates (25,26). Hydrogenase activity was induced anaerobically in the dark, and H 2 photoproduction was monitored the following day. Rates of photosynthesis, respiration, and H 2 photoproduction in liquid cultures were determined as described previously (27).
Hydrogen was assayed from the head space of anaerobically sealed cultures using a Varian model 3700 gas chromatograph. For the methyl viologen (MV) 1 assay of hydrogenase activity, cells were removed and added to an equal volume of anaerobic 2ϫ MV solution (10 mM MV (oxidized), 25 mM potassium phosphate, pH 6.9, and 0.2% Triton X-100) in a sealed anaerobic vial. Degassed sodium dithionite was added to a final concentration of 4 mM to initiate H 2 production from reduced MV.
Anaerobic Induction of Liquid Cell Suspensions-C. reinhardtii cultures were grown on Tris acetate-phosphate medium to ϳ20 g/ml total chlorophyll, centrifuged at 2500 ϫ g for 5 min, and resuspended in one-tenth volume of AIB induction buffer containing 50 mM potassium phosphate, pH 7, and 3 mM MgCl 2 (27). Samples were placed in vials that were wrapped with aluminum foil to exclude light, sealed with a rubber septum, flushed with argon for 15 min, and then incubated anaerobically in the dark at room temperature.
Southern and Northern Blot Analyses-Southern blotting experiments were performed using standard methodology. Genomic DNA was extracted and purified using a DNeasy Plant Mini kit (Qiagen). Northern blot analysis was performed using 10 g of total RNA for each sample as described previously (20). Probes were labeled using [␣-32 P]dCTP (ICN) and the Rediprime II DNA random prime labeling system (Amersham Biosciences).
Western Blot Analysis-After 4 h of anaerobic induction, cells were lysed under anaerobic conditions. Aerobic control samples were lysed immediately after resuspension in AIB buffer. Cells were disrupted with gentle rocking in lysis buffer (50 mM Tris, pH 8.5, and 0.25% Triton X-100) for 30 min, and the cellular extract was centrifuged for 10 min at 10,000 ϫ g. The hydrogenase protein was partially purified from induced and noninduced cells under strictly anaerobic conditions by loading the lysed supernatant containing the hydrogenase activity onto a Q-Sepharose Fast Flow column (Amersham Biosciences). The column was washed once with 2 column volumes of wash buffer (50 mM Tris, pH 8.5, and 100 mM KCl) and eluted with 2 column volumes of elution buffer (50 mM Tris, pH8.5, and 250 mM KCl). Approximately 85% of the hydrogenase activity detected in the crude lysate from induced WT cultures was recovered in the partially purified fraction. Protein samples were concentrated using an Amicon protein concentration cell and a YM10 membrane. Equal amounts of protein (A 280 ) were loaded and separated using standard SDS-PAGE methodologies. Western blotting was performed using a Bio-Rad Mini-Protean III electrophoresis and blotting apparatus as described in the manufacturer's instructions. The primary hydrogenase antibody was derived from a synthetic peptide (DKAKRQAALYNL) containing a sequence common to both the HydA1 protein and the HydA2 protein and was generated commercially in rabbits (Sigma Genosys). The secondary antibody was obtained commercially as an alkaline phosphatase conjugate (Bio-Rad), and standard chemochromic detection techniques were utilized for hydrogenase detection.
Gene Identification-DNA regions flanking the insertion site of pJD67 were determined using genome walking. DNA downstream of the insertion site was amplified using the PCR methods outlined for the Universal GenomeWalker kit and the Advantage-GC Genomic PCR mix, both from Clontech. Coding sequences for both the HydEF and HydG proteins were obtained by sequencing the cDNA corresponding to both genes. The cDNA constructs were obtained from the Kazusa DNA Research Institute (www.kazusa.or.jp/). All DNA products were sequenced by the University of California, Davis sequencing facility.
Complementation-A bacterial artificial chromosome clone containing the HydEF and HydG genes was obtained from the Clemson University Genetics center. The genomic HydEF gene was obtained by KpnI digestion of the bacterial artificial chromosome clone, and the insert containing the full-length HydEF gene with promoter and termination sequences was cloned into the KpnI site of pSP124S (a gift from Saul Purton, University College, London). The resulting plasmid, pMP101, contains the HydEF gene and the Ble r gene used for antibiotic selection. The pMP101 plasmid was linearized by digestion with SwaI and transformed into the hydEF-1 mutant using the glass bead method of Kindle (28). Controls, using cells only or 1 g of pSP124S (29), were also used.
Heterologous Expression and Purification-Expression of active C. reinhardtii HydA1 was achieved by cloning the HydEF and HydG cDNA constructs into E. coli expression plasmids driven by the T7 promoter. The two genes were cloned into the pACYC Duet expression plasmid (Novagen). Additional control plasmids containing only the HydEF or the HydG gene were also cloned into the pACYC plasmid. The C. reinhardtii HydA1 gene was cloned into pETBlue-1, and a Strep-Tactin affinity tag (Strep-Tag II) was added to its C terminus for the affinity purification of HydA1. Plasmids were co-transformed into E. coli Bl-21 (DE3) cells (Novagen). The presence of appropriate plasmids was verified by restriction analysis and sequencing. Expression and purification of tagged HydA1 was done as follows. An inoculum from an overnight culture of transformed BL21 (DE3) cells was grown in L-broth containing the appropriate antibiotics. Cells were grown until the A 600 reached 0.5-0.7, and isopropyl-␤-D-thiogalactopyranoside (Novagen) was added to 1 mM. After a 1-h aerobic induction, cultures were made anaerobic by purging with argon for 5 h. Cells were harvested and then disrupted on ice by sonication. HydA1-StrepTag II was purified using Strep-Tactin-Sepharose (IBA) according to the manufacturer's instructions and assayed for hydrogenase activity using MV.

Mutant
Characterization-In C. reinhardtii, two [Fe] hydrogenase enzymes, HydA1 and HydA2, have been reported (19,20). To identify genes required for the expression and activity of these enzymes, we used chemochromic H 2 sensors (26) to screen a random insertional mutagenesis library for clones incapable of photoproducing H 2 following the required anaerobic induction. Mutants were generated by transforming the Arg7 gene into C. reinhardtii strain CC425, which is an arginine auxotroph. The Arg7 gene is randomly incorporated into the C. reinhardtii genome and disrupts small sections of wildtype (WT) genomic DNA. The mutant hydEF-1 was identified by its inability to produce detectable quantities of H 2 , as shown in Fig. 1A. The dark blue spots (Fig. 1A) observed for the other five colonies from the same library are indicative of WT H 2 production capacity.
The mutant hydEF-1 grew on minimal medium agar plates with CO 2 as the sole carbon source, demonstrating that the cells were photosynthetically competent. Furthermore, photosynthetic and respiratory rates of both the parental and hydEF-1 strains were measured in liquid media using a Clarktype electrode (Fig. 1B). Compared with the WT, the hydEF-1 mutant exhibited normal rates of respiration and photosynthetic O 2 evolution. This demonstrates that the lack of H 2 photoproduction activity in hydEF-1 is not the consequence of a secondary metabolic or photosynthetic electron transport defect but rather is specific to the hydrogenase enzyme.
Hydrogenase activity in C. reinhardtii is induced by anaerobiosis (4,5,20,30,31). This is achieved either in the dark by using an inert gas (or exogenous reductant) to purge O 2 from sealed cultures or in the light by depriving sealed cultures of sulfur, which results in attenuated rates of photosynthetic O 2 evolution (4, 5, 32). Hydrogen production, following dark anaerobic induction, was monitored from WT and hydEF-1 mutant cultures using several techniques as follows: 1) assaying the initial rates of H 2 photoproduction (Fig. 1C) with a Clarktype electrode; 2) using gas chromatography to detect hydrogenase activity mediated by reduced MV; and 3) analyzing fermentative H 2 production with gas chromatography. In contrast to the case with WT cultures, H 2 production was not detected from hydEF-1 mutant cultures by any of these assays. Moreover, hydEF-1 mutant cultures that were induced anaerobically in the light under conditions of sulfur deprivation failed to produce any detectable H 2 . One-liter CC425 WT cultures consistently produced at least 70 ml of H 2 over the course of several days under identical conditions (data not shown). We therefore concluded that the hydEF-1 mutant is unable to synthesize an active [Fe] hydrogenase under any of our induction and assay conditions.
Identification of the HydEF and HydG Genes-To determine the genetic mutation responsible for the observed phenotype of the hydEF-1 mutant, we cloned and sequenced the genomic DNA flanking the mutagenizing Arg7 insert using a genome walking strategy. The gene disrupted by Arg7 insertion was determined by comparing the flanking WT sequence to the recently sequenced C. reinhardtii genome. The deleted gene in hydEF-1, denoted HydEF, was shown to encode a protein with two unique domains. The N-terminal portion of the HydEF Genomic DNA was digested with NcoI, blotted, and probed using a DNA sequence that had been deleted in the hydEF-1 mutant.
[Fe] Hydrogenase Assembly Proteins protein is homologous to a distinct group of proteins that, to date, are only found in prokaryotes containing [Fe] hydrogenases and belongs to a previously uncharacterized subset of the radical S-adenosylmethionine (designated "Radical SAM") protein superfamily (33). The C-terminal portion of the HydEF protein contains a domain with predicted GTPase activity. This domain is homologous to a second distinct group of prokaryotic proteins, which are also unique to organisms that contain [Fe] hydrogenases. Directly adjacent to the disrupted HydEF gene in C. reinhardtii is a second gene, HydG, which is arranged in an order suggestive of divergent expression from the same promoter region. BLAST searches revealed that proteins homologous to HydG comprise a third set of unique proteins that also belong to the Radical SAM protein superfamily. As with HydE and HydF, the HydG homologues are only found in prokaryotes with [Fe] hydrogenases. The cDNAs corresponding to HydEF and HydG in C. reinhardtii were obtained and then sequenced to confirm the protein coding sequence of the two genes. A schematic indicating the genomic organization of the C. reinhardtii genes and the site of HydEF disruption is shown in Fig. 2A.
Strikingly, in the genomes of Bacteroides thetaiotaomicron, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Shewanella oneidensis the HydE, HydF, and HydG genes form putative operons with [Fe] hydrogenase structural genes (Fig.  3). However, the functions of HydE, HydF, and HydG have not, until now, been assigned. As discussed below, our data indicate that these proteins are required for the assembly of active [Fe] hydrogenase, and, therefore, we have named the C. reinhardtii genes HydEF and HydG in accordance with the suggested hydrogenase nomenclature (2). In C. reinhardtii, the HydEF gene is assigned the two letters E and F to correspond to the two distinct genes observed in prokaryotic organisms. Fig. 3 compares the C. reinhardtii HydEF and HydG protein homologies to prokaryotic organisms containing [Fe] hydrogenases. Fig. 3 also shows the organization of the HydE, HydF, and HydG open reading frames in relationship to the putative [Fe] hydrogenase gene(s) within these organisms. Although the proposed [Fe] hydrogenase assembly genes observed in the previously mentioned organisms are found in putative operons along with the [Fe] hydrogenase structural genes, these proposed assembly proteins within the majority of the organisms shown in Fig. 3 are found separated from the structural genes.
Complementation of the HydEF Gene-To link the observed loss of H 2 production in the C. reinhardtii hydEF-1 mutant to the disruption of HydEF, we used gene complementation. Genomic DNA containing the WT HydEF gene was obtained from a single bacterial artificial chromosome clone found in a library of C. reinhardtii genomic DNA. The bacterial artificial chromosome plasmid containing the HydEF gene was digested with appropriate restriction enzymes to generate a fragment predicted to contain only the full-length HydEF genomic gene and its putative promoter. This insert was cloned into plasmid SP124S, which contains the Ble gene that confers resistance to [Fe] Hydrogenase Assembly Proteins the antibiotic zeocin. The hydEF-1 mutant was transformed with this construct, grown on Tris acetate-phosphate agar plates containing zeocin, and clones with restored H 2 -production capacity were obtained as shown in Fig. 2B. Integration of the complementing gene and verification of the mutant background were confirmed by Southern blotting (Fig. 2C). The CC425 sample shows the WT band, which is absent in both the mutant and the complemented clone. The complemented clone shows two strong bands corresponding to multiple random integration of the transformed HydEF genomic fragment into the mutant genome as well as a faint band that may represent integration of only a portion of the HydEF gene.
Analysis of Gene Expression and [Fe] Hydrogenase Accumulation-Northern blot analyses were then performed to determine whether the observed loss of hydrogenase activity in the hydEF-1 mutant was due to disruption of HydA1 and/or HydA2 gene transcription and whether HydEF and HydG are co-expressed anaerobically with the hydrogenase genes. RNA aliquots were collected from aerobic WT and hydEF-1 mutant cultures as well as from WT and hydEF-1 mutant cultures anaerobically induced in the dark for 0.5 and 4 h. Fig. 4, A-D compare, respectively, the expression profiles of the HydA1, HydA2, HydG, and HydEF genes from both CC425 parental WT and hydEF-1 mutant cultures. The data demonstrate that HydEF and HydG are anaerobically induced concomitantly with the HydA1 and HydA2 genes in WT cultures. Likewise, the HydA1, HydA2, and HydG transcripts are also induced anaerobically in the hydEF-1 mutant, and, as expected, the HydEF transcript is absent. The presence of HydA1 and HydA2 transcripts in anaerobically induced hydEF-1 cultures clearly indicates that disruption of the HydEF gene does not affect hydrogenase transcription in any significant fashion and that the loss of H 2 production in hydEF-1 cultures is not the consequence of a defect in hydrogenase gene transcription.
Western blots were then obtained to determine the consequence of HydEF gene disruption on hydrogenase protein levels (Fig. 4E). An antibody designed to recognize both C. reinhardtii HydA1 and HydA2 was used to probe for the presence of hydrogenase proteins. As expected, the partially purified WT sample (see "Experimental Procedures") shows only a single anaerobically induced band with an electrophoretic mobility of ϳ47-48 kDa due to co-migration of the two hydrogenases. Although full-length HydA1 and HydA2 hydrogenase enzymes from C. reinhardtii have predicted masses of 53.1 and 53.7 kDa, respectively, HydA1 undergoes N-terminal proteolytic processing of a chloroplast transit peptide sequence, resulting in a mature 47.5-kDa protein localized in the chloroplast (31). The HydA2 protein is predicted to undergo similar processing, resulting in an estimated 47.3-kDa mature protein (20). The Western data from anaerobically induced hydEF-1 cultures indicate that an immunologically detectable enzyme is also found in hydEF-1 mutant cultures despite the lack of detectable enzyme activity. The electrophoretic mobility of the hydrogenase band from hydEF-1 mutant cultures is shifted slightly lower relative to the WT band and is consistent with the electrophoretic mobility of unprocessed C. reinhardtii hydrogenase. In the case of [NiFe] hydrogenases, proteolytic processing occurs after insertion of nickel, resulting in shifted Western bands relative to the unprocessed [NiFe]-enzyme (34,35). The presence of shifted bands in the anaerobically induced hydEF-1 protein extracts suggests that such processing might also occur in the case of C. reinhardtii [Fe] hydrogenases lacking a fully assembled active site.
Heterologous Expression of C. reinhardtii HydA1 in E. coli-Additional evidence supporting the conclusion that the HydEF and HydG proteins are required for the formation of an active [Fe] hydrogenase is shown by the heterologous expression of an active C. reinhardtii HydA1 protein in E. coli, a bacterium that lacks a native [Fe] hydrogenase. The HydA1 protein was expressed as a fusion protein containing a Strep-Tag II affinity sequence and purified from E. coli extracts. The expression of the HydA1 construct alone or co-expression of the HydA1 and HydEF or HydA1 and HydG genes in E. coli all resulted in the expression of non-functional HydA1 protein after purification, as shown in Fig. 5. However, the co-expression of C. reinhardtii  2) and hydEF-1 mutant (lanes 3 and 4) samples. The blot was probed with an antibody designed to recognize both C. reinhardtii HydA1 and C. reinhardtii HydA2.
FIG. 5. Hydrogen production rates from purified HydA1 heterologously expressed in E. coli either alone or co-expressed with the indicated Hyd proteins. Hydrogen production was measured using the methyl viologen-based assay. The data shown represent the average of four independent experiments; average deviations from the mean are shown.

[Fe] Hydrogenase Assembly Proteins
HydA1 along with both HydEF and HydG in anaerobic E. coli cultures yielded an active HydA1 enzyme (Fig. 5). Because the expression system has yet to be optimized, the amount of active HydA1 obtained from independent experiments is rather low and varies significantly. Nevertheless, functional [Fe] hydrogenase was only obtained in the presence of all three expressed genes. It should be noted that some Radical SAM proteins act with extremely low turnover numbers and may even be reactants and not catalysts (33). DISCUSSION Radical SAM Homology-The HydEF and HydG proteins belong to the Radical SAM (also known as the AdoMet radical) superfamily. These proteins participate in numerous biochem-  [Fe] Hydrogenase Assembly Proteins 25716 ical reactions including but not limited to sulfur insertion, radical formation, organic ring synthesis, and anaerobic oxidation (33,36). The HydG protein and the HydE domain of the C. reinhardtii HydEF protein both contain the signature Cys-X 3 -Cys-X 2 -Cys motif that is typically found within the Radical SAM protein superfamily (Fig. 6). This motif coordinates a redox active [4Fe4S] cluster under reducing conditions (33, 36 -38). The reactions performed by Radical SAM proteins are typically initiated by the generation of a free radical after the reductive cleavage of S-adenosylmethionine at the [4Fe4S] cluster, which yields methionine and a 5Ј-deoxyadenosyl radical (36,37,39). This high-energy organic radical then abstracts FIG. 6-continued [Fe] Hydrogenase Assembly Proteins 25717 a hydrogen atom from substrates unique to each Radical SAM protein (33). Postulated Roles for HydEF and HydG in H-cluster Assembly-Radical SAM proteins are frequently involved in the anaerobic synthesis of complex biomolecules and coordinate unusual [FeS] clusters that are often labile (33,36,38,40,41). These characteristics are consistent with the types of chemistries required to synthesize the unique ligands of the H-cluster and to assemble the [Fe] hydrogenase catalytic cluster. A recent classification of the Radical SAM superfamily suggests that the most distantly related proteins, including biotin synthase (BioB) and the nitrogenase accessory protein NifB, appear to be involved in sulfur transfer (33). Remarkably, iron and sulfur that originate from the metabolic product of NifB, the NifB-cofactor, ultimately become incorporated into the [FeMo] cofactor of dinitrogenase (42), another enzyme capable of H 2 production. Thus, there is precedent for the involvement of a Radical SAM protein in the donation of iron to the catalytic metal cluster of an [Fe]-metalloenzyme, and we propose that the HydE and/or HydG proteins described here play a similar role in the mobilization of iron for assembly of the [Fe] hydrogenase H-cluster.
The H-cluster also requires CN, CO, and the putative di(thiomethyl)amine ligand. It is conceivable that the accessory proteins HydEF and/or HydG described here are also responsible for the biosynthesis and assembly of these products coordinated to iron. Because CN and CO are among the most toxic compounds in biology and likely do not exist freely within the cell, it would be necessary to synthesize these ligands at the site of H-cluster assembly. In the case of the [NiFe] hydroge-FIG. 6-continued [Fe] Hydrogenase Assembly Proteins nases, strong evidence indicates that CN and CO are synthesized by the HypE and HypF proteins using carbamoyl phosphate as a precursor to form a thiocarbamate (43,44). However, no homologues of the HypE and HypF proteins have been observed in C. reinhardtii or in other organisms containing only [Fe] hydrogenases (2). This suggests an alternative pathway for CN and CO synthesis or an alternative means to form thiocarbamate. Radical SAM proteins utilize chemistries that include organic radical formation, persulfide formation, pyroxidal phosphate activation, thiocarbonyl formation, and amine migration (33,36,45), all or any one of which could be involved in the synthesis of the H-cluster organic ligands.
Homology alignments between C. reinhardtii HydEF and HydG relative to their prokaryotic homologues are shown in Fig. 6. In addition to the Radical SAM motifs, the HydG and HydF proteins have other conserved sequences with the potential to coordinate metal ions. These include a E(A/G)CXH and a (I/V)HC(G/A)(G/A)C motif near the C terminus of the HydF domain and a CT(A/G)CYR motif near the C terminus of the HydG protein. All three of these motifs are strictly conserved in the [Fe] hydrogenase assembly proteins, but they are absent from other Radical SAM proteins, which suggests that these motifs are unique to the [Fe] hydrogenase accessory proteins. Several other conserved amino acids are found throughout the HydEF and HydG proteins; however, the elucidation of roles for these determinants and the potential metal binding motifs in the assembly of [Fe] hydrogenase will have to await future investigation. It should also be noted that the HydF domain of the HydEF protein contains a putative GTPase domain, and the HypB protein, which also has GTPase activity, facilitates nickel incorporation into the active site of [NiFe] hydrogenases. Interestingly, neither the HydEF nor the HydG proteins are highly homologous to the TM1420 protein characterized from T. maritima (23). The latter is only 8.5 kDa long and does not contain a characteristic Radical SAM motif. This suggests that TM1420 may be unique to T. maritima, which has the most complex [Fe] hydrogenase characterized to date.
Heterologous Expression-The heterologous expression of C. reinhardtii HydA1 in E. coli demonstrates that only two C. reinhardtii gene products, HydEF and HydG (equivalent to three prokaryotic genes) are required for the assembly of HydA1; however, a minimum of seven accessory gene products are required for the formation of an active [NiFe] hydrogenase enzyme (7). This is consistent with the prediction that the [Fe] hydrogenases may require fewer maturation proteins because these enzymes lack nickel (2). The existence of entirely unique maturation proteins required for the assembly of [Fe] hydrogenase is consistent with the absence of a phylogenetic relationship between [NiFe] and [Fe] hydrogenases.
Previous attempts to express the CpI or DdH [Fe] hydrogenase enzymes in E. coli resulted in the synthesis of inactive proteins that were unable to evolve or uptake H 2 gas (46,47). In contrast, transformation of the cyanobacterium Synechococcus PCC7942 with the CpI [Fe] hydrogenase structural gene yielded strains that expressed an active [Fe] hydrogenase (47). Given that there is no biochemical or genetic evidence for the presence of an [Fe] hydrogenase in Synechoccocus PCC7942, the authors postulated that accessory proteins responsible for assembling the Synechococcus [NiFe] hydrogenases are flexible enough to also activate the CpI [Fe] hydrogenase enzyme. It is not clear why this is possible in Synechoccocus and not in E. coli, but these results emphasize the complex nature of hydrogenase expression and activation in different microorganisms.
In summary, two novel genes found in C. reinhardtii, HydEF and HydG, are strictly conserved in organisms containing [Fe] hydrogenases. The HydEF and HydG genes are transcribed anaerobically in parallel with the HydA1 and HydA2 [Fe] hydrogenase genes in C. reinhardtii. Disruption of HydEF abolishes all H 2 production, and, although full-length hydrogenase protein is detected by Western blotting, no enzyme activity is observed. Hydrogen production is restored after complementation of the hydEF-1 mutant with WT genomic DNA containing the HydEF gene. Moreover, we report the first successful coexpression of the C. reinhardtii HydEF, HydG, and HydA1 genes in E. coli and the synthesis of an active [Fe] hydrogenase in this bacterium. The current study also identifies a new class of metallo-enzyme accessory proteins and assigns assembly function to two proteins belonging to a subset of the Radical SAM superfamily. Characterization of these [Fe] hydrogenase assembly proteins will greatly facilitate additional examination of the mechanism by which [Fe] hydrogenases are synthesized in nature.