With a redox potential of around -420 mV for the H2/2H+ couple, hydrogen is an ideal electron donor to drive redox processes in biological systems. Equally, proton reduction to hydrogen can be used as a means of removing excess reducing equivalents from biological systems generated through oxidation of reduced carbon substrates. In contrast to protons, hydrogen diffuses readily across biological membranes, and this forms a simple means of generating a proton gradient (1). These properties of hydrogen have meant that this molecule has been, and remains to this day, a major driver for sustaining the redox processes that govern life on earth.
The term hydrogenase (Hyd; EC 18.104.22.168) was introduced by Stephenson and Stickland (2) to describe the enzyme that catalyses the reversible oxidation of molecular hydrogen to protons and electrons. Hydrogenases are ubiquitous and found in all three domains of life (3, 4). Phylogenetic analysis of the primary structure of the catalytic subunit revealed that Hyd enzymes have evolved as three independent classes (5) further emphasizing the significance of hydrogen metabolism for the evolution of life on our planet. These three classes use different combinations of metal ion cofactors to activate hydrogen and have been named based on the metals found in their respective catalytic center. They include the [NiFe]-Hyd found in bacteria and archaea, [FeFe]-Hyd found in eukarya and bacteria and [Fe]-Hyd, so far found only in certain methanogenic archaea (3, 4). All of these enzymes have in common a strict requirement for iron, and this metal is essential for catalysis. In the [NiFe]-Hyd and [FeFe]-Hyd classes, iron is also necessary for electron transfer processes within these enzymes.
Frequently, [NiFe]-Hyd are modular heteromeric enzymes comprising a large subunit, where catalysis occurs, and a small subunit, which relays electrons to and from the active site (Figure 1). The bimetallic active site is buried deeply within the large subunit to which molecular hydrogen diffuses through specific gas channels (6–8). This metal center catalyzes the endergonic (200 kJ mol-1 at 20°C), heterolytic bond cleavage into a proton and a hydride ion (9). The two electrons derived from proton release are channeled through the small subunit via a series of electron carriers, which are usually iron-sulfur (Fe-S) clusters (Figure 1), to specific physiological electron acceptors (4, 6). Occasionally, the Hyd is linked to a diaphorase module to facilitate hydrogen-coupled NAD+ reduction (10), or the Hyd can be membrane-associated through a specialized membrane anchor module bearing either Fe-S clusters or cytochromes b, which relays the electrons to and from the quinone pool (11).
The first structural analysis of a [NiFe]-Hyd (12) revealed the presence of diatomic ligands associated with the Fe ion in the active site. This finding immediately drew attention to an infrared study (13) in which evidence was presented for the association of the diatomic ligands CO and CN- with [NiFe]-Hyd. Meanwhile, it has been clearly demonstrated that all [NiFe]-Hyd enzymes characterized, to date, have one CO and two CN- ligands associated with the Fe ion (14, 15). These diatomic ligands maintain the Fe in a low-spin and low-redox state to facilitate hydrogen activation at the nickel ion. Remarkably, both CO and CN- are also key ligands to the Fe ions in the active site of [FeFe]-Hyd, while [Fe]-Hyd also has CO liganded to the Fe ion but has a pyridinol group located in the vicinity of the iron, instead of the cyanide ligand (4). These enzymes thus represent a remarkably lucid example of convergent evolution.
From the foregoing, it is immediately apparent that iron is an essential component of all Hyd enzymes. While key aspects of nickel uptake and delivery for active site biosynthesis of the [NiFe]-Hyd enzymes is comparatively well understood [for reviews see (15–19)], considerably less is known regarding the biochemical routes taken by iron and how its specific incorporation into the metal centers of these enzymes occurs. Therefore, this review will focus on our current knowledge regarding biosynthesis of the metal centers in [NiFe]-Hyd with a particular emphasis on what has recently been revealed about the incorporation of iron into the hydrogenases of Escherichia coli.
Organization and structure of the metal centers in [NiFe]-hydrogenase
Despite its location deep within the large subunit, the bimetallic active site of [NiFe]-Hyd is within 12 Å of the proximal Fe-S cluster in the small subunit (Figure 1), thus allowing facile electron transfer. Usually, the medial and distal Fe-S cluster named with respect to their positions relative to the active site, are also located at appropriate distances to one another to enable efficient electron transfer between them (Figure 1). With a size in the range of 60–65 kDa, and due to the comparatively large contact surface between the subunits (7, 12), the large subunit has no need for its own Fe-S cluster to mediate electron transfer to the small subunit.
The composition and organization of the Fe-S clusters in the hydrogenase small subunits vary, often depending on the environmental habitat of the microbe that synthesizes the hydrogenase, as well as whether the hydrogenase is involved in energy conservation NAD+ reduction, or in dissipation of redox equivalents through hydrogen evolution. While the oxygen-sensitive, soluble, periplasmic hydrogen-oxidizing hydrogenases of Desulfovibrio species have proximal and distal [4Fe-4S] clusters with a high-potential medial [3Fe-4S] cluster (20), the more oxygen-tolerant, membrane-associated hydrogen-oxidizing hydrogenases of the Knallgas bacterium Ralstonia eutropha (also known as Cupriavidus necator), and the obligate autotrophic γ proteobacterium Hydrogenovibrio marinus, have recently been shown to have a novel, proximal [4Fe-3S] cluster along with a medial [3Fe-4S] cluster and a distal [4Fe-4S] cluster (21, 22). The oxygen-tolerant, membrane-associated hydrogen-oxidizing Hyd-1 of the facultative anaerobe E. coli and Hyd-5 of Salmonella enterica also has this complement of Fe-S clusters in its small subunit (Figure 1) (23–25). Coordination of the [4Fe-3S] cluster is achieved by two extra Cys residues that are not necessary for activity but for conferring oxygen tolerance (26, 27). There is convincing evidence that the novel [4Fe-3S] cluster has a function in ‘protecting’ the [NiFe] active site from oxygen attack by avoiding the slowly reacting and ‘unready’ Ni-A state and instead adopting only the rapidly activating and ‘ready’ Ni-B state, in which the Fe-S clusters are electron-rich and thus better able to resist an oxygen attack on the active site (23, 26, 27). Indeed, recent evidence also suggests that the [4Fe-3S] cluster functions together with the high-potential medial [3Fe-4S] to facilitate the direct two-electron reduction of oxygen to water (24).
[NiFe]-Hyd enzymes involved in hydrogen evolution, such as Hyd-3 of E. coli, have a small subunit predicted to carry a single [4Fe-4S] cluster (Figure 2) (28, 29). Similarly, the HoxY small subunit of the bidirectional Hyd of cyanobacteria is predicted to have a single [4Fe-4S] cluster (30).
Ni-Fe(CN)2CO biosynthesis and maturation of the large subunit
The ability to purify a soluble HycE subunit in the absence of further proteins of the associated formate hydrogenlyase complex (FHL) (Figure 2) led to the establishment of a maturation model of [NiFe]-Hyd by Böck and colleagues (15, 18). The term maturation encompasses both the biosynthesis and the insertion of the [NiFe] site, and this requires a specific set of accessory maturation proteins, which have been termed Hyp (hydrogenase pleiotropic) because a mutation in any one of the key hyp genes results in a strain devoid of active [NiFe]-Hyd (15, 18). The core Hyp proteins are HypC, HypD, HypE, and HypF, and together, these proteins synthesize the Fe(CN)2CO center (Figure 3) (15, 31). The HypA and HypB proteins are required for delivery and insertion of the Ni2+ ion subsequent to the insertion of the completed Fe(CN)2CO moiety into the active site of the large subunit. Frequently, paralogs of certain Hyp proteins are found, particularly if the microorganism in question synthesizes multiple [NiFe]-Hyd. Thus, for example, mutations in hypC and hypA prevent maturation of Hyd-3 in E. coli but not the maturation of Hyd-1 or Hyd-2. This is because proteins homologous to HypC and HypA, which are required for Hyd-1 and Hyd-2 maturation, are encoded by the hybG and hybF genes, respectively (Figure 3) (32, 33).
Origin and assembly of the diatomic ligands on the Fe atom
Pioneering studies by the group of Böck demonstrated that the accessory proteins HypC, D, E, and F form a complex, which ultimately assembles the iron atom with its diatomic ligands, and this is inserted into HycE, the catalytic subunit of Hyd-3 (34). It is assumed that this mechanism is universal, with perhaps slight modifications, for all microbes synthesizing [NiFe]-Hyd.
One of the substrates required for diatomic ligand formation is carbamoyl phosphate, which is derived metabolically from citrulline and which has been shown by labeling experiments with 13C-citrulline to be exclusively the source of the two CN- ligands (35, 36). The first step in the generation of the CN- ligand involves the activation of carbamoyl phosphate by HypF, which adenylates and therefore stabilizes the carbamoyl group (37). Using the energy derived from hydrolysis of a further ATP HypF transfers the carbamoyl moiety from carbamoyladenylate onto the C-terminal cysteine (Cys-322) of HypE generating a thiocarboxamide (38–40). The HypE accessory protein then catalyzes the ATP-dependent dehydration of the thiocarboxamide to a thiocyanate, which is thus activated for transfer onto an iron atom (37–40). Through the use of a carAB mutant (devoid of carbamoyl phosphate synthetase), it was clearly demonstrated that neither the CN- nor the CO ligands were transferred to the HypCD scaffold complex (see also below), despite only CN- ligand synthesis being impaired (41). This suggests CN- ligand biosynthesis precedes the synthesis and incorporation of the CO ligand, although it still cannot be ruled out that the addition of the CO and CN- moieties could be codependent.
While E. coli cells grown in the presence of exogenously supplied 13CO gas inserted the ligand quantitatively into the active site of Hyd-2 or heterologously synthesized regulatory hydrogenase from R. eutropha (35, 36, 42), it could be demonstrated unequivocally that the concentration of the gas in the atmosphere was insufficient for it to be the direct source of the ligand (35, 36, 42). Therefore, the precursor of the CO ligand must have a metabolic origin. For heterotrophically growing R. eutropha, labeled 1,3-13C2-glycerol was shown to serve as an indirect source for the CO ligand to the regulatory hydrogenase; however, with the multitude of metabolic pathways glycerol can enter, it remains unclear what the immediate precursor is (42).
The iron atom for preassembly of the FeCN2CO moiety is bound to HypD, but its [4Fe-4S] cluster can be ruled out as a donor (43, 44). Instead, the crystal structure of a HypC and HypD complex (45) suggests that Cys-2 of HypC and Cys-41 of HypD (Cys-38HypD in the structure from Thermococcus kodakaraensis) coordinate an Fe ion; however, no iron was observed in the structure presumably because it was labile in the presence of oxygen. Indeed, recent studies have established that strictly anaerobic conditions are necessary to isolate the HypCD complex carrying the Fe(CN)2CO moiety (45). Notably, this anaerobic complex carries up to two additional iron ions, apart from those in the [4Fe-4S] cluster, suggesting that one of these additional iron ions coordinates the diatomic ligands (46).
Binding studies performed in the presence of oxygen confirmed that a complex between HypC and HypD was able to bind iron between the highly conserved Cys-2 of HypC and Cys-41 (Cys-38 in T. kodakaraensis) of HypD, while neither HypC nor HypD alone could interact with the metal (45). More recently, anaerobically isolated E. coli HypC and HybG proteins were shown to contain substoichiometric amounts of iron (47). Isolation of either protein aerobically revealed no iron association, confirming the oxygen-labile nature of the bound iron moiety. Moreover, amino acid variants lacking Cys-2 also failed to bind iron, even when the protein was isolated anaerobically confirming that Cys-2 is essential for coordination of the iron ion (47).
Infrared spectroscopy enables visualization of CN-- and CO-stretching frequencies when these ligands are bound to the Fe atom in the active site (13), and these signals can also be detected on a HypCD complex (see model in Figure 4) (31, 41). A closer examination of the IR spectrum of the anaerobically isolated HypCD complex revealed an additional signature, which could be correlated with the stretching frequency associated with iron-bound CO2 (46). This observation immediately suggested CO2 as the possible direct precursor of CO, possibly being reduced by electrons channeled through the Fe-S cluster of HypD. Moreover, although HypD in the absence of HypC can bind the Fe(CN)2CO cofactor stably, no evidence for bound CO2 was obtained via IR spectroscopy, and only maximally, five iron ions could be identified associated with the protein (31), suggesting perhaps that a key role for HypC might be the delivery of both the iron and CO2 to the HypCD complex. Recent studies, indeed, confirmed that anaerobically isolated HypC and HybG proteins carry CO2 bound to iron (47). Further experiments are, however, required to demonstrate that both the iron and the CO2 bound by HypC are the direct precursors of the corresponding Fe ion and CO ligand in the Fe(CN)2CO moiety. Moreover, exogenously supplied 13C-hydrogen carbonate could not be incorporated as a CO ligand to hydrogenase by E. coli ΔcarAB cells (36), while 13C-acetate (labeled at the α-carbon) resulted in labeled CO ligands in Allochromatium vinosum (48). This would be commensurate with the earlier proposal that the source of the CO ligand might be endogenously produced CO2 (48). Thus, a HypC interaction partner that delivers CO2 needs to be identified.
Intermediate scaffold complexes in the biosynthesis of oxygen-tolerant hydrogenases
Early studies on the hydrogenase system in the endosymbiotic bacterium Rhizobium leguminosarum led to the identification of HupK, which shares common structural motifs with the large subunit of [NiFe]-hydrogenases (49). While not a hydrogenase per se, HupK was insightfully proposed to have a scaffold function during maturation. More recent studies in a number of bacteria, including R. eutropha (50) and Thiocapsa roseopersicina (51), which synthesize aerotolerant hydrogenases, have indeed confirmed a scaffold function for HupK. Moreover, these bacteria typically also synthesize at least one additional HypC paralog, which interacts with the HupK homolog and with the precursor form of the large subunit. In R. leguminosarum, this HypC homolog, termed HupF (52), carries a C-terminal extension, which confers upon the protein the ability to protect the hydrogenase large subunit from the deleterious effects of oxygen during maturation. A model based on work in R. eutropha was proposed, in which a complex comprising HoxV (homolog of HupK) and HoxL (homolog of HupF) accept the oxygen-labile Fe(CN)2CO moiety from the HypCD complex and deliver it to the apo-form of the hydrogenase large subunit (50).
Closure of the active site
The insertion of the Fe(CN)2CO has been clearly shown to precede the insertion of the nickel ion into the large subunit (53–55). In order to insert the nickel ion into the hydrogenase large subunit, which has been shown most clearly for Hyd-3 in E. coli, the proteins HypA, HypB, and SlyD are required, whereby the HypB GTPase dimerizes in a GTP-dependent manner to transfer the nickel ion, while HypA serves as a scaffold protein (Figure 4) (56–58). SlyD is a peptidyl-prolyl cis/trans isomerase, and although not essential for hydrogenase maturation, it facilitates the release and transfer of the Ni2+ ion as well as mediating the interaction of HypB with HycE (59, 60). Because a hypB mutant can be phenotypically complemented through the addition of high amounts of nickel to the growth medium (61), this indicates that the HypAB-SlyD nickel-insertion machinery has a kinetic role in nickel insertion. In bacteria such as Helicobacter pylori, HypA and HypB are also responsible for delivery and insertion of the nickel ion into the active site of urease. Notably, H. pylori mutants lacking the hypAB genes have reduced pathogenicity (19, 62).
Completion of hydrogenase large subunit maturation [an exception being the regulatory hydrogenase of R. eutropha (63)] requires the proteolytic removal of a C-terminal peptide [reviewed in (15)]. The sequence and length of these C-terminally cleaved peptides vary between hydrogenases, and each large subunit has its own highly specific endoprotease (15). Proteolytic processing of the large subunit by the endoprotease likely leads to a conformational change that encloses the active site after its delivery by HypC (Figure 5) (64). The sequence of the C-terminal peptide is not highly conserved, probably reflecting the requirement for a specific endoprotease for each hydrogenase, but its presence is essential for nickel insertion. Proteolysis does not happen in the absence of nickel, and therefore, the metal is a key determinant that governs protease function (65–69). The specific endoproteases for Hyd-1 and Hyd-2 are HyaD and HybD, respectively (Figure 3) (70). As proteolytic processing only occurs when nickel, and no other divalent cation (68, 71), is present, this indicates that whenever processing is observed, a bona fide Ni-Fe(CN)2CO active site is present (Figure 5A). It has been observed that in the absence of this proteolytic processing event, or in the absence of the large subunit, the small subunit is rapidly degraded (72). This indicates a tight coupling between large and small subunit processing.
Fe-S cluster biosynthesis
Iron-sulphur proteins have various functions ranging from regulation of gene expression to structural and electron-transferring roles. The modular design of oxidoreductases shows an array of Fe-S clusters in their small subunits separated by less than ≈14 Å (73), which is shown in Figure 1 for hydrogenases. To ensure efficient electron transfer, some large modular oxidoreductase, such as formate dehydrogenases and nitrate reductases, require an additional Fe-S cluster in their large subunit (73, 74). Assembly and insertion of Fe-S clusters are not spontaneous processes, but there is also not a dedicated protein for every single Fe-S cluster type. Instead, three general and independent machineries for biosynthesis of Fe-S clusters have evolved (75, 76). Studies on nitrogenase biosynthesis in Azotobacter vinelandii identified the NifU and NifS proteins, which have a key role in Fe-S and FeMo cofactor biosynthesis (77–79). A further set of proteins referred to as the Isc (iron sulfur cluster) machinery was subsequently discovered to be encoded on the genome of A. vinelandii (75, 76). Meanwhile, the Isc system has been discovered in numerous microorganisms, including E. coli, together with the orthologous Suf (sulfur assimilation) Isc biosynthetic machinery (76, 80, 81). The Suf system is comprised of the six gene products of the sufABCDSE operon, which is mainly expressed under oxidative stress (82). The isc operon, on the other hand, is negatively regulated by its own regulator IscR and comprises the genes iscRSUA-hscBA-fdx-iscX (83, 84). Both systems have homologous components. Sulfur is recruited from cysteine by the desulfurases IscS or SufS and then assembled into an unstable [2Fe-2S] cluster on the scaffolds IscU or SufBCD, respectively (75, 85). The physiological iron source, although unknown, can be supplied in vitro in the form of free Fe2+ or can be delivered on the frataxin-like protein CyaY (Figure 4). The potential role of CyaY in vivo has only been suggested so far through the demonstration of an interaction between CyaY and IscS, as well as through its in vivo regulatory role (86–89). Transfer of the completed Fe-S cluster to the apo-protein target is probably accomplished by the A-type carrier (ATC) proteins IscA, SufA, and ErpA, whose functions have been studied through a combination of mutagenesis, protein-protein interaction, and in vitro Fe-S cluster transfer studies (90). It has been shown that for certain key anaerobic respiratory enzymes, the Suf system plays no significant role, while the Isc system is crucial for the generation of active respiratory hydrogenases (72, 74). Strains devoid of the IscA, ErpA, or IscU proteins lack activity of Hyd-1 and Hyd-2, and this is mainly due to the absence of the small subunits (72). Notably, in iscA and erpA mutants, the large subunits of these enzymes are nevertheless processed, indicating that the Isc system is not solely responsible for active site iron insertion (Figure 5A) (72).
The activity of the FHL complex was shown to be reduced when the A-type carrier (ATC) proteins were missing, but was absent only when the gene coding for the scaffold protein IscU or when both the iscA and erpA genes were deleted (91). The latter observation indicates partial redundancy of function between IscA and ErpA. The level of the respective Hyd-1 and Hyd-2 large subunits was significantly reduced in the isc mutants (Figure 5A). This suggests either that enhanced degradation of the large subunit in the absence of matured small subunit occurred or possibly that expression of the hya and hyb operons was reduced due to an incompletely functional Isc system. It has been shown that the IscR regulator represses hya and hyb operon expression under aerobic conditions (92, 93), and deletion of the iscR gene causes derepression of hya and hyb expression (94). Nevertheless, it has been demonstrated recently (95, 96) that both apo-IscR and [2Fe-2S]-IscR have different regulatory functions, and because the presence of the Fe-S cluster in IscR is determined by the activity of the Isc machinery, any mutation affecting Isc function has a knock-on effect on IscR-regulated genes. Future experiments will be required to determine to what extent mutations in isc operon genes regulate expression of operons encoding anaerobic oxidoreductases.
In a recent study, it was revealed that the Isc system is also able to provide basic [4Fe-4S] clusters during the heterologous expression of [FeFe]-Hyd in the absence of their specific maturases (97, 98). Moreover, apparently, the Isc system can be forced to erroneously introduce an oxygen labile Fe-S cluster into the active sites of HyaB and HybC, the large subunits of the [NiFe]-hydrogenases Hyd-1 and Hyd-2, respectively. This cluster is coordinated by the four cysteinyl residues that normally ligand the [NiFe]-cofactor (99) and appears to be introduced when the maturation machinery cannot insert the [NiFe]-cofactor, thus possibly serving to stabilize the protein during maturation. These observations raise the intriguing question as to how the erroneous insertion of Fe-S clusters is prevented, and indeed, what governs delivery and construction of the highly diverse Fe-S clusters like those found in the small subunits?
Subunit assembly and membrane transport: the checkpoints
Generally, H2-uptake Hyd that couple H2 oxidation to energy conservation are membrane-associated with their catalytic large subunits located in the periplasm. This necessitates translocation of the mature enzyme across the membrane by the twin-arginine translocation (Tat) machinery (100, 101). In contrast, Hyd enzymes located in the cytoplasm (see Hyd-3 in Figure 2) often catalyze H2 production, and these do not rely on the Tat machinery for the completion of their assembly (11).
The H2-oxidizing Hyd-1 of E. coli is encoded by the hyaABCDEF operon. The first gene, hyaA, encodes the small Fe-S cluster containing subunit, while the second gene, hyaB, encodes the catalytic subunit, which harbors the [NiFe]-active site (Figure 3) (102). The HyaA, but not the HyaB, polypeptide carries an N-terminal Tat signal sequence, and this is responsible for translocation of the HyaA-HyaB heterodimer (or possibly dimer of dimers) across the membrane via a ‘piggy-back’ mechanism (103). Similarly, the first gene of the hybOABCDEFG operon encodes the small subunit of Hyd-2, and HybO also has a Tat signal sequence (Figure 3) (104, 105). The small subunits of both enzymes are also anchored in the membrane via a hydrophobic C-terminal α-helix; however, electrons are transferred to the quinone pool via a further membrane-integral subunit, which harbors a b-type heme cofactor in the case of Hyd-1 (HyaC). Unusually, in the case of Hyd-2, the membrane anchor HybB lacks any apparent cofactor (Figure 2) (11, 106). However, Hyd-2, which has an additional ferredoxin-like subunit that has an N-terminal Tat signal peptide, is required for electron transfer to other membrane-associated oxidoreductases and appears to be translocated across the membrane independently of the HybO-HybC complex (106).
Owing to their order in the operon, the genes encoding the small subunits of the H2-oxidizing Hyds are transcribed and translated prior to those of the large subunits. However, if the maturation of the large subunit is not completed, the Tat signal peptide of the small subunit is not removed, the small subunit protein is rapidly degraded, and the unprocessed large subunit remains in the cytoplasm (Figure 5) (72). If the Fe-S cluster of the small subunit cannot be introduced due to a defective Isc machinery, the small subunit is also rapidly degraded, and the large subunit also remains in the cytoplasm; notably, the maturation of the large subunit does not depend on whether maturation of the small subunit occurs or not (72). Thus, the maturation of the large subunit must be completed first and only then is the maturation of the small subunit completed, presumably concomitant with transport through the Tat translocon. It is likely that the interaction of the complex with the Tat system also controls protein turnover of the small subunits, and a system must be in place that ‘masks’ the Tat signal peptide from the Tat machinery until the complex is ready for transport. Private chaperones in E. coli specifically bind the small subunit precursor, and they are encoded by the genes hyaE, hyaF, and hybE in the operons of the respective H2-oxidizing Hyd (Figure 3). Homologs of HyaE and HyaF, called HupG and HupH, respectively, were shown to be essential for maturation of the small subunit of hydrogenase in R. leguminosarum (107), while in R. eutropha, the HyaE homolog, HoxO, interacts with the signal peptide of the small subunit HoxK (108).
The structure of HyaE has been determined, and it belongs to a class of proteins with a thioredoxin fold; however, they lack the Cys residues necessary for redox reactions (109). Notably, a hoxO mutant of R. eutropha fails to synthesize active membrane-bound Hyd, and the mutation caused the precursor of the large subunit (HoxG) to be delivered prematurely to the membrane, while processed HoxG was detectable in the cytoplasm (110). This suggests that the HyaE/HoxO proteins coordinate maturation of the small subunit after large subunit processing has occurred and deliver the completed complex to the Tat machinery.
In E. coli, single hyaE or hyaF deletions have no apparent defects in respiratory hydrogenase assembly and membrane targeting (106); however, when both gene products are absent, no Hyd-1 activity is observed indicating that some redundancy in function between the proteins exists (102). The R. eutropha HyaF homolog HoxQ influences large subunit maturation, and it was suggested that it is necessary for other key steps of small subunit maturation (110), while in R. leguminosarum, the HupH protein (homolog of HyaF in E. coli) was shown to interact specifically with the premature small subunit (107). Owing to the fact that the hydrogenases of R. eutropha are synthesized under aerobic conditions, it is likely that the bacterium has further chaperones, e.g., HoxR and HoxT, which protect the Fe-S cluster in the small subunit from oxygen damage. Indeed, it was shown that the rubredoxin HoxR plays a role in establishing the final complement of Fe-S clusters in the small subunit (111, 112). Such a function is unnecessary in E. coli because the hydrogenases are only synthesized under anaerobic conditions, and this explains why no homologs of HoxR and HoxT exist in E. coli.
The HybE protein was shown to recognize specifically the Tat signal sequence of the small subunit HybO as well as the C-terminal extension of the large subunit HybC (113). The protein is not required for maturation of the large subunit HybC but for coordinated transport of both subunits to the membrane, as in ΔhybE strains, the small subunit was found in the membrane without the catalytic partner HybC (103).
A dual role for these accessory proteins in the modification of the proximal [4Fe-3S] cluster has been suggested (25) as well as in protection against reactive oxygen species (112) (Figure 4). Possibly, these hydrogenase-specific accessory proteins, together with the local amino acid sequence of the small subunit protein, determine the type of cluster that is inserted. Using model peptides mimicking the local amino acid structure of an Fe-S protein, it has been shown that amino acid exchanges influence the occurrence of particular Fe-S cluster types (114).
In contrast to these findings, the cytoplasmically oriented FHL complex is more tolerant toward defects in Fe-S cluster biosynthesis (91). Unfortunately, little is known about the assembly of the FHL complex and the stoichiometry of its components, except that none of the subunits harbors a signal sequence for membrane transport. It makes sense that only correctly inserted Fe-S cluster-containing proteins are allowed to pass through the Tat machinery, while FHL proteins can partially assemble prior to processing (C.P. and F. Sargent, unpublished data).
Uptake of metals and regulation
The uptake of metal ions from the medium is highly specific in microorganisms and independent of the concentration of other metals in the medium (115). The most abundant metals of the 343 cytoplasmic metal proteins in Pyrococcus furiosus are iron and zinc (97%) followed by tungsten and nickel (<2.5%) with <0.5% of other metals (115). Transport and insertion of the nickel ion into the active site of Hyd has been well investigated. A specific nickel ABC (ATP binding cassette) transporter was discovered through mutagenesis studies, and mutations that prevent its synthesis reduce [NiFe]-Hyd activity. Nevertheless, this reduction in hydrogenase activity can be phenotypically complemented through the addition of high amounts of nickel to the growth medium, indicating nonspecific uptake of nickel via other transport systems (17, 116, 117). Expression of the nik genes encoding the nickel transporter is Fnr dependent and is repressed by the nickel-responsive transcription factor NikR in the presence of high intracellular nickel (17, 117, 118). Fnr has an oxygen-labile Fe-S cluster, and it ensures that, in E. coli, the nik operon is only expressed anaerobically when the hydrogenases are synthesized (Figure 4).
While the Nik ABC transporter is the only transporter specific for nickel uptake in E. coli, an additional nickel permease is present in H. pylori (119). R. leguminosarum also encodes a nickel permease, HupE (120), while a homolog, HoxN, transports nickel by a uniport mechanism in R. eutropha (121). Intriguingly, Salmonella enterica has an aerobically synthesized Hyd enzyme, which contains a fully functional [NiFe]-active site ((122); C.P. and F. Sargent, unpublished results), suggesting that either Ni2+ uptake occurs aerobically, or sufficient stores of the ion are available to satisfy aerobic enzyme biosynthesis.
The mechanisms governing iron uptake are much more diverse in E. coli. There are high-affinity uptake systems for ferrous iron (FeoABC), various siderophore-mediated uptake systems (FecA-E, FepA-G, FhuA-D), and further uptake systems that transport iron nonspecifically (123–125). Iron homeostasis is controlled by the iron-responsive transcription factor Fur (ferric uptake regulator) (126). Fur binds iron, thus, enabling it to bind to a consensus sequence in the -35 region of promoters of iron-responsive genes repressing transcription (127). Surprisingly, a fur deletion strain of E. coli shows reduced FHL activity due to lower transcription levels of the hyc genes, while transcription of the genes coding for the H2-uptake Hyd remains unaffected (128). An inverse effect is observed when iron uptake cannot be accomplished. Strains defective in the Feo system have drastically reduced Hyd-1 and Hyd-2 levels with only small amounts of large subunit detectable, while Hyd-3, and thus FHL, levels are less significantly affected (Figure 4) (129). Only further deletion of genes coding for the iron citrate transporter, Fec, and genes of the enterochelin biosynthesis pathway cause a reduction in FHL activity, revealing a distinct bias for maintaining different Hyd activities within the cell under specific conditions (129).
[NiFe]-hydrogenases are evolutionarily ancient enzymes. Examination of their active sites reveals that all components derive from inorganic compounds abundant on early earth (130) including nickel and iron sulfides, carbon monoxide, and cyanide. Conceivably, somehow these components formed an early primitive catalyst that was able to generate hydrogen in the reducing and acidic environment or to activate volcanic hydrogen gas; evolution has retained and adapted these compounds as key catalytic components of modern-day hydrogenase. Iron remains the key component, and the evolution of a specific set of accessory proteins has facilitated the biosynthesis of these iron-based cofactors from metabolic intermediates as well as their assembly into the appropriate apo-protein ‘cage’, which considerably enhances their catalytic efficiency and assures stability by protecting them from the bulk phase. One future challenge will be to design equally efficient catalysts that are considerably more robust toward aqueous environments.
Recent collaborative efforts between chemists, biochemists, spectroscopists, and biologists (24, 131, 132) have highlighted the rapid advances that can be achieved in understanding the chemistry and biology of hydrogen activation by combining expertise. The challenge for the future will be to develop these synergies further to provide a complete understanding of [NiFe]-hydrogenase biosynthesis. Much new and surprising biochemistry awaits discovery in this important scientific area.
What is becoming increasingly apparent is that biosynthesis of cofactors takes place on assembly platforms or scaffolds often comprising numerous accessory proteins (31, 41, 89, 133). Each of these accessory proteins probably has one or more specific biochemical function, but each is also important for the integrity and functionality of the complex. Moreover, there are components of this platform that have the job of specifically recognizing the appropriate apo-protein target for cofactor delivery. The private chaperones of apo-protein targets presumably have the role of ‘masking’ peptide sequences, e.g., binding the Tat signal peptide to prevent premature translocation of an ‘immature’ protein through the Tat machine. But these chaperones might also act as guides, or docking sites, for cofactor delivery proteins, ensuring the correct cofactor is delivered to the correct site at the appropriate time. Considering the fact that the cytoplasmic protein concentration is likely to be ≥250 mg/ml (134), it is probable that the maturation of complex metalloenzymes such as [NiFe]-hydrogenases occurs on large multiprotein complexes that include all necessary components. These complexes are likely to be highly localized, possibly membrane-associated, particularly for those that are translocated across the cytoplasmic membrane by the Tat translocon. Further development of high-resolution cell biological methods will facilitate elucidation of these complexes and their cellular localization. The development of X-ray free-electron laser technology (135), single-molecule studies together with methods such as high-resolution secondary ion mass spectrometry (nano-SIMS) (136) will resolve the structures of these complexes, the physical interactions between components, and the individual biochemical steps on these biosynthetic pathways.
With knowledge gained from the structural and spectroscopic analyses of hydrogenases, it will be possible to make major advances in constructing robust and highly efficient hydrogenase biomimics (131, 132), greatly facilitating the development of hydrogen-driven fuel cell technology as one of several future alternative clean energy sources.
[NiFe]-hydrogenases harbor Fe in different types of Fe-S clusters and in the active site, which also has a nickel ion.
The Fe-S clusters are inserted by the Isc system; however, the mechanism underlying the insertion and modification of the clusters is unknown.
Active site iron is decorated with CO and CN- diatomic ligands on a maturation protein scaffold complex comprising HypC, HypD, HypE, and HypF.
Iron and CO2 are bound by the HypC protein and might represent intermediates in active site biosynthesis.
The Feo transport system is the main delivery route of iron for [NiFe]-hydrogenase cofactor biosynthesis in E. coli.
Iron metabolism not only influences hydrogenase activity by determining cofactor availability but also affects transcription of the hydrogenase structural operons.
In E. coli, assembly of the H2 uptake hydrogenases is more strictly controlled than that of the cytoplasmic hydrogen-evolving FHL complex, possibly reflecting differential iron requirements, allowing metabolic demands to be met.
There are several key assembly checkpoints during hydrogenase biosynthesis, which, if not correctly passed, result in subunit turnover.
This work was supported by the Deutsche Forschungsgemeinschaft (Sa 343/3-2) and by EFRE funds of the EU to RGS and grant BB/I02008X/1 from the Biotechnology and Biological Sciences Research Council to Prof. Frank Sargent in whose laboratory C.P. currently works.
Conflict of interest statement
The authors declare that they have no competing interests.
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About the article
Constanze Pinske was born in Berlin, Germany, in 1983. From 2003 to 2008, she studied Biochemistry at the Martin-Luther University Halle-Wittenberg, Germany. She did her studies for her Diploma (2008) and PhD thesis (2012) on the topic of ‘[NiFe]-hydrogenase maturation in Escherichia coli’ with Gary Sawers in the division of General Microbiology, MLU Halle. She moved to Dundee Scotland in 2012 where she is working on the biochemistry of the formate hydrogenlyase complex as a postdoc with Frank Sargent.
R. Gary Sawers
Gary Sawers was born in Edinburgh, Scotland, in 1959. Having studied Biochemistry at Heriot-Watt University, Edinburgh, from 1977 to 1881, he moved to the University of Dundee, Scotland, where he undertook PhD studies on the analysis of the membrane-associated [NiFe]-hydrogenases of Escherichia coli. In 1985, he moved to the Ludwig-Maximillians University of Munich, Germany, where he worked in the laboratory of August Böck studying molecular aspects of bacterial fermentation. From 1995 until 2005, he was a project leader in the Department of Molecular Microbiology at the John Innes Center, Norwich UK. From 2006, he worked at the Max-Planck Institute for Terrestrial Microbiology with Rudolf Thauer on the biochemistry of hydrogenase and moved in August 2007 to take up the chair of General Microbiology at the Martin-Luther University Halle-Wittenberg, Germany.
Published Online: 2014-03-01
Published in Print: 2014-03-01