Solution structure of Escherichia coli HypC

Escherichia coli HypC plays an important role in the maturation process of the pre-maturated HycE, the large subunit of hydrogenase 3. It serves as an iron transfer as well as a chaperone protein during the maturation process of pre-HycE, and interacts with both HypD and HycE. The N-terminal cysteine residue of HypC plays a key role in the protein-protein interactions. Here, we present the three-dimensional structure of E. coli HypC, the first solution structure of HupF/HypC family. Our result demonstrates that E. coli HypC consists of a typical OB-fold beta-barrel with two C-terminal helixes. Sequence alignment and structural comparison reveal that the hydrophobic region on the surface of E. coli HypC, as well as the highly flexible C-terminal helixes, may involve in the interactions of E. coli HypC with other proteins.     

Maturation of [NiFe]-hydrogenases in Escherichia coli: the HypC cycle

Carbamoyl phosphate (CP) has been implicated as an educt for the synthesis of the CO and CN ligands of the metal centre of [NiFe]-hydrogenases in Escherichia coli, since CP synthetase mutants (carAB) are unable to generate active hydrogenases due to a block in enzyme maturation. Citrulline, when added to the growth medium in high concentrations, compensated for the phenotype of the mutants. It is now shown that overexpression of the argI gene lowered the effective concentration of citrulline, thus proving that the amino acid serves as a source for CP. The DeltaCarAB mutant accumulated a complex consisting of the hydrogenase maturation proteins HypC and HypD. This complex was resolved upon citrulline addition and followed-up by the appearance of a complex between HypC and the precursor of the large subunit of hydrogenase 3, preHycE. In the absence of the hycE gene, the HypC-HypD complex did not disappear upon addition of citrulline but developed into a form migrating slower in a non-denaturing polyacrylamide gel, providing strong evidence for the notion that the HypC-HypD complex is the intermediate in hydrogenase maturation where CP or its products are added to the iron atom of the metal centre. This step precedes nickel insertion, since extracts of carAB cells that had been cultivated in the absence of citrulline are unable to process preHycE after the addition of nickel. Complex formation between HypC and HypD, and between HypC and preHycE display dependence on identical primary structure elements of HypC. On the basis of the results, a cycle of HypC activity is proposed whose function is to transfer the iron atom that has been liganded at the HypC-HypD complex to the precursor of the large hydrogenase subunit.     

Interplay between the specific chaperone-like proteins HybG and HypC in maturation of hydrogenases 1, 2, and 3 from Escherichia coli

The hybG gene product from Escherichia coli has been identified as a chaperone-like protein acting in the maturation of hydrogenases 1 and 2. It was shown that HybG forms a complex with the precursor of the large subunit of hydrogenase 2. As with HypC, which is the chaperone-like protein involved in hydrogenase 3 maturation, the N-terminal cysteine residue is crucial for complex formation. Introduction of a deletion into hybG abolished the generation of active hydrogenase 2 but only quantitatively reduced hydrogenase 1 activity since HypC could replace HybG in this function. In contrast, HybG could not take over the role of HypC in a DeltahypC genetic background. Overproduction of HybG, especially of the variants with the replaced N-terminal cysteine residue, strongly interfered with hydrogenase 3 maturation, apparently by titrating some other component(s) of the maturation machinery. The results indicate that the three hydrogenase isoenzymes not only are interacting at the functional level but are also interconnected during the maturation process.     

NiFe hydrogenase active site biosynthesis: identification of Hyp protein complexes in Ralstonia eutropha

Biosynthesis of the NiFe hydrogenase active site is a complex process involving the action of the Hyp proteins: HypA-HypF. Here we investigate the mechanism of NiFe site biosynthesis in Ralstonia eutropha by examining the interactions between HypC, HypD, HypE, and HypF1. Using an affinity purification procedure based on the Strep-tag II, we purified HypC and HypE from different genetic backgrounds as complexes with other hydrogenase-related proteins and characterized them using immunological analysis. Copurification of HypC and HoxH, the active site-containing subunit of the soluble hydrogenase in R. eutropha, from several different genetic backgrounds suggests that this complex forms early in the maturation process. With respect to the Hyp proteins, it is shown that HypE and HypF1 formed a stable complex both in vivo and in vitro. Furthermore, HypC and HypD functioned as a unit. Together, they were able to interact with HypE to form a range of complexes probably varying in stoichiometry. The HypC/HypD/HypE complexes did not involve HypF1 but appeared to be more stable when HypF1 was also present in the cells. We hypothesize that HypF1 is able to modify some component of the HypC/HypD/HypE complex. Since we have also seen that HypF1 and HypE form a complex, it is likely that HypF1 modifies HypE. On the basis of these results, we propose a complete catalytic cycle for HypE. First, it is modified by HypF1, and then it can form a complex with HypC/HypD. This activated HypE/HypC/HypD complex could then decompose by donating active site components to the immature hydrogenase and regenerate unmodified HypE.     

The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]-hydrogenases

Carbamoylphosphate has been shown to be the educt for the synthesis of the CN ligands of the NiFe metal centre of hydrogenases from Escherichia coli. In the absence of carbamoylphosphate, cells accumulate a complex of two hydrogenase maturation proteins, namely HypC and HypD for the synthesis of hydrogenase 3. A procedure for the purification of wild-type HypD protein or of a biologically active derivative carrying the Strep-tagII((R)) at the N terminus has been developed. HypD is a monomeric protein possessing about 4 mol of iron per mol of protein. Electron paramagnetic resonance (EPR) and Mossbauer spectroscopy demonstrated that the iron is present as a diamagnetic [4Fe-4S](2+) cluster. The complex between HypC and HypD can be cross-linked by a number of thiol and primary amine-specific linkers. When HypD and HypC were overproduced side-by-side with HypE, the HypC-HypD complex contained substoichiometric amounts of HypE whose proportion in the complex could be augmented when HypF was also overproduced. HypE trapped in this complex could be carbamoylated by protein HypF and after dehydration transferred the cyano group to the HypC-HypD part of the complex. Free HypC and HypD were not cyanated by HypE-CN. An active HypC-HypD complex from anaerobic cells was inactivated by incubation with K(3)[Fe(CN)(6)] but not with K(4)[Fe(CN)(6)]. The results suggest the existence of a dynamic complex between the hydrogenase maturation proteins HypD, HypC, HypE and HypF, which is the site of ligand biosynthesis and attachment to the iron atom of the NiFe site in hydrogenase 3.     

Analysis of the HypC-hycE complex, a key intermediate in the assembly of the metal center of the Escherichia coli hydrogenase 3

The formation of a complex between the specific chaperone-type protein HypC and the precursor form of the large subunit HycE in the maturation pathway of hydrogenase 3 from Escherichia coli has been studied by targeted replacement of amino acids in both proteins. HypC and its homologs contain the motif MC(L/I/V)(G/A)(L/I/V)P at the amino terminus, from which the methionine residue is post-translationally removed. The exchange of the cysteine residue led to complete loss of the ability to interact with the precursor form of HycE, but replacement of the proline residue had no effect. Site-directed replacement of the conserved cysteine residues in HycE involved in nickel binding was also performed. Exchange of Cys(241) resulted in the inability of the HycE variant to interact with HypC and to incorporate nickel. The variants of HycE in which Cys(244) and Cys(531) were replaced by alanine residues were unable to incorporate nickel, although the mutated proteins could interact with HypC. Intriguingly, the precursor of HycE in which the Cys(534) residue was exchanged could form the complex with HypC, could incorporate nickel, and was C-terminally processed, but it delivered an inactive enzyme. Our findings are in favor of a model in which binding of HypC masks Cys(241); Cys(244) and Cys(531) bind the iron and nickel moieties, respectively; and C534 closes the bridge between the two metals after C-terminal processing has taken place.     

The role of the active site-coordinating cysteine residues in the maturation of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16

The H₂-splitting active site of [NiFe] hydrogenases is tightly bound to the protein matrix via four conserved cysteine residues. In this study, the nickel-binding cysteine residues of HoxC, the large subunit of the H₂-sensing regulatory hydrogenase (RH) from Ralstonia eutropha, were replaced by serine. All four mutant proteins, C60S, C63S, C479S, and C482S, were inactive both in H₂ sensing and H₂ oxidation and did not adopt the native oligomeric structure of the RH. Nickel was bound only to the C482S derivative. The assembly of the [NiFe] active site is a complex process that requires the function of at least six accessory proteins. Among these proteins, HypC has been shown to act as a chaperone for the large subunit during the maturation process. Immunoblot analysis revealed the presence of a strong RH-dependent HypC-specific complex in extracts containing the C60S, C63S, and C482S derivatives, pointing to a block in maturation for these mutant proteins. The lack of this complex in the extract containing C479S indicates that this specific cysteine residue might be crucial for the interaction between HoxC and HypC.This work is dedicated to Prof. H.G. Schlegel on the occasion of his 80th birthday.     

Protein interactions and localization of the Escherichia coli accessory protein HypA during nickel insertion to [NiFe] hydrogenase

Nickel delivery during maturation of Escherichia coli [NiFe] hydrogenase 3 includes the accessory proteins HypA, HypB, and SlyD. Although the isolated proteins have been characterized, little is known about how they interact with each other and the hydrogenase 3 large subunit, HycE. In this study the complexes of HypA and HycE were investigated after modification with the Strep-tag II. Multiprotein complexes containing HypA, HypB, SlyD, and HycE were observed, consistent with the assembly of a single nickel insertion cluster. An interaction between HypA and HycE did not require the other nickel insertion proteins, but HypB was not found with the large subunit in the absence of HypA. The HypA-HycE complex was not detected in the absence of the HypC or HypD proteins, involved in the preceding iron insertion step, and this interaction is enhanced by nickel brought into the cell by the NikABCDE membrane transporter. Furthermore, without the hydrogenase 1, 2, and 3 large subunits, complexes between HypA, HypB, and SlyD were observed. These results support the hypothesis that HypA acts as a scaffold for assembly of the nickel insertion proteins with the hydrogenase precursor protein after delivery of the iron center. At different stages of the hydrogenase maturation process, HypA was observed at or near the cell membrane by using fluorescence confocal microscopy, as was HycE, suggesting membrane localization of the nickel insertion event.     

Maturation of Rhizobium leguminosarum Hydrogenase in the Presence of Oxygen Requires the Interaction of the Chaperone HypC and the Scaffolding Protein HupK

[NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)₂CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)₂CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼12 μm O₂) and bacteroids from legume nodules (∼10–100 nm O₂). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen.     

A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit

The large subunit HoxC of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.     

Chaperones specific for the membrane-bound [NiFe]-hydrogenase interact with the Tat signal peptide of the small subunit precursor in Ralstonia eutropha H16

Periplasmic membrane-bound [NiFe]-hydrogenases undergo a complex maturation pathway, including cofactor incorporation, subunit assembly, and finally twin-arginine-dependent membrane translocation (Tat). In this study, the role of the two accessory proteins HoxO and HoxQ in the maturation of the membrane-bound [NiFe]-hydrogenase (MBH) of Ralstonia eutropha H16 was investigated. MBH activity was absent in soluble as well as membrane fractions of cells with deletions in the respective genes. The absence of HoxO and HoxQ led to degradation of the small subunit precursor (preHoxK) of the MBH. The two accessory proteins directly interacted with preHoxK prior to assembly of active MBH dimer in the cytoplasm. MBH mutants with modified Tat signal peptides were disrupted in preHoxK/HoxO/HoxQ complex formation. Isolated HoxO and HoxQ proteins formed a complex in vitro with the chemically synthesized HoxK Tat signal peptide. Two functions of the two chaperones are discussed: (i) protection of the Fe-S cluster containing HoxK subunit under oxygenic conditions, and (ii) avoidance of HoxK export prior to dimerization with the large MBH subunit HoxG.     

Crystal structures of [NiFe] hydrogenase maturation proteins HypC, HypD, and HypE: insights into cyanation reaction by thiol redox signaling

[NiFe] hydrogenase maturation proteins HypC, HypD, and HypE catalyze the insertion and cyanation of the iron center of [NiFe] hydrogenases by an unknown mechanism. We have determined the crystal structures of HypC, HypD, and HypE from Thermococcus kodakaraensis KOD1 at 1.8 A, 2.07 A, and 1.55 A resolution, respectively. The structure of HypD reveals its probable iron binding and active sites for cyanation. An extended conformation of each conserved motif of HypC and HypE allows the essential cysteine residues of both proteins to interact with the active site of HypD. Furthermore, the C-terminal tail of HypE is shown to exist in an ATP-dependent dynamic equilibrium between outward and inward conformations. Unexpectedly, the [4Fe-4S] cluster environment of HypD is quite similar to that of ferredoxin:thioredoxin reductase (FTR), indicating the existence of a redox cascade similar to the FTR system. These results suggest a cyanation reaction mechanism via unique thiol redox signaling in the HypCDE complex.     

Delivery of iron-sulfur clusters to the hydrogen-oxidizing [NiFe]-hydrogenases in Escherichia coli requires the A-type carrier proteins ErpA and IscA

During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements.     

Involvement of hyp Gene Products in Maturation of the H2-Sensing [NiFe] Hydrogenase of Ralstonia eutropha

The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and HypX for assembly of the H(2)-activating [NiFe] site. In this study we examined the maturation of the regulatory hydrogenase (RH) of Ralstonia eutropha. The RH is a H(2)-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [NiFe] hydrogenases. Here we demonstrate that the RH regulatory activity was barely affected by mutations in hypA, hypB, hypC, and hypX and was not substantially diminished in hypD- and hypE-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of (63)Ni into the RH from overproducing cells revealed that the assembly of the [NiFe] active site is dependent on all Hyp functions, with the exception of HypX. We conclude that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) is involved in an efficient incorporation of the [NiFe] center into the RH.     

Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina.

There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon-based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane-bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T. roseopersicina.     

Network of hydrogenase maturation in Escherichia coli: role of accessory proteins HypA and HybF

We have studied the roles of the auxiliary protein HypA and of its homolog HybF in hydrogenase maturation. A mutation in hypA leads to the nearly complete blockade of maturation solely of hydrogenase 3 whereas a lesion in hybF drastically but not totally reduces maturation and activity of isoenzymes 1 and 2. The residual level of matured enzymes in the hybF mutant was shown to be due to the function of HypA; HybF, conversely, was responsible for a minimal residual activity of hydrogenase 3 in the mutant hypA strain. Accordingly, a hypA DeltahybF double mutant was completely blocked in the maturation process. However, the inclusion of high nickel concentrations in the medium could restore limited activity of all three hydrogenases. The results of this study and of previous work (M. Blokesch, A. Magalon, and A. B?ck, J. Bacteriol. 189:2817-2822, 2001) show that the maturation of the three functional hydrogenases from Escherichia coli is intimately connected via the activity of proteins HypA and HypC and of their homologs HybF and HybG, respectively. The results also support the suggestion of Olson et al. (J. W. Olson, N. S. Mehta, and R. J. Maier, Mol. Microbiol. 39:176-182, 2001) that HypA cooperates with HypB in the insertion of nickel into the precursor of the large hydrogenase subunit. Whereas HypA is predominantly involved in the maturation of hydrogenase 3, HybF takes over its function in the maturation of isoenzymes 1 and 2.     

Transcription profiles of hydrogenases related genes in the cyanobacterium Lyngbya majuscula CCAP 1446/4

Background  

Lyngbya majuscula CCAP 1446/4 is a N₂-fixing filamentous nonheterocystous strain that contains two NiFe-hydrogenases: an uptake (encoded by hupSL) and a bidirectional enzyme (encoded by hoxEFUYH). The biosynthesis/maturation of NiFe-hydrogenases is a complex process requiring several accessory proteins for e.g. for the incorporation of metals and ligands in the active center (large subunit), and the insertion of the FeS clusters (small subunit). The last step in the maturation of the large subunit is the cleavage of a C-terminal peptide from its precursor by a specific endopeptidase. Subsequently, the mature large and small subunits can assemble forming a functional enzyme.     

Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3′-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.     

Biogenesis of a respiratory complex is orchestrated by a single accessory protein

The biogenesis of respiratory complexes is a multistep process that requires finely tuned coordination of subunit assembly, metal cofactor insertion, and membrane-anchoring events. The dissimilatory nitrate reductase of the bacterial anaerobic respiratory chain is a membrane-bound heterotrimeric complex nitrate reductase A (NarGHI) carrying no less than eight redox centers. Here, we identified different stable folding assembly intermediates of the nitrate reductase complex and analyzed their redox cofactor contents using electron paramagnetic resonance spectroscopy. Upon the absence of the accessory protein NarJ, a global defect in metal incorporation was revealed. In addition to the molybdenum cofactor, we show that NarJ is required for specific insertion of the proximal iron-sulfur cluster (FS0) within the soluble nitrate reductase (NarGH) catalytic dimer. Further, we establish that NarJ ensures complete maturation of the b-type cytochrome subunit NarI by a proper timing for membrane anchoring of the NarGH complex. Our findings demonstrate that NarJ has a multifunctional role by orchestrating both the maturation and the assembly steps.