RESUMO
[NiFe]-hydrogenases (Hyds) comprise a small and a large subunit. The latter harbors the biologically unique [NiFe](CN)2CO active-site cofactor. The maturation process includes the assembly of the [Fe](CN)2CO cofactor precursor, nickel binding, endoproteolytic cleavage of the large subunit, and dimerization with the small subunit to yield active enzyme. The biosynthesis of the [Fe](CN)2CO moiety of [NiFe]-Hyd-1 and Hyd-2 occurs on the scaffold complex HybG-HypD (GD), whereas the HypC-HypD complex is specific for the assembly of Hyd-3. The metabolic source and the route for delivering iron to the active site remain unclear. To investigate the maturation process of O2-tolerant Hyd-1 from Escherichia coli, we developed an enzymatic in vitro reconstitution system that allows for the synthesis of Hyd-1 using only purified components. Together with this in vitro reconstitution system, we employed biochemical analyses, infrared spectroscopy (attenuated total reflection FTIR), mass spectrometry (MS), and microscale thermophoresis to monitor the iron transfer during the maturation process and to understand how the [Fe](CN)2CO cofactor precursor is ultimately incorporated into the large subunit. We demonstrate the direct transfer of iron from 57Fe-labeled GD complex to the large subunit of Hyd-1. Our data reveal that the GD complex exclusively interacts with the large subunit of Hyd-1 and Hyd-2 but not with the large subunit of Hyd-3. Furthermore, we show that the presence of iron in the active site is a prerequisite for nickel insertion. Taken together, these findings reveal how the [Fe](CN)2CO cofactor precursor is transferred and incorporated into the active site of [NiFe]-Hyd.
Assuntos
Proteínas de Escherichia coli , Hidrogenase , Ferro , Chaperonas Moleculares , Oxirredutases , Transporte Biológico , Domínio Catalítico , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Hidrogenase/química , Hidrogenase/metabolismo , Ferro/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Oxirredutases/química , Oxirredutases/metabolismoRESUMO
The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN- and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN- and CO ligands on the scaffold complex HypCD.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas/metabolismo , Enxofre/metabolismo , Monóxido de Carbono/metabolismo , Domínio Catalítico , Dissulfetos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Elétrons , Escherichia coli/genética , Íons/metabolismo , Ligantes , Oxirredução , Ligação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Espectroscopia de Mossbauer/métodosRESUMO
Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H2). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN)2CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN)2CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase.
Assuntos
Coenzimas/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Hidrogenase/metabolismo , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Carboxil e Carbamoil Transferases/química , Carboxil e Carbamoil Transferases/genética , Carboxil e Carbamoil Transferases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dimerização , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Hidrogenase/química , Hidrogenase/genética , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Dobramento de Proteína , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Rapid disulfide bond formation and cleavage is an essential mechanism of life. Using large amplitude Fourier transformed alternating current voltammetry (FTacV) we have measured previously uncharacterized disulfide bond redox chemistry in Escherichia coli HypD. This protein is representative of a class of assembly proteins that play an essential role in the biosynthesis of the active site of [NiFe]-hydrogenases, a family of H2-activating enzymes. Compared to conventional electrochemical methods, the advantages of the FTacV technique are the high resolution of the faradaic signal in the higher order harmonics and the fact that a single electrochemical experiment contains all the data needed to estimate the (very fast) electron transfer rates (both rate constants ≥ 4000 s-1) and quantify the energetics of the cysteine disulfide redox-reaction (reversible potentials for both processes approximately -0.21 ± 0.01 V vs SHE at pH 6). Previously, deriving such data depended on an inefficient manual trial-and-error approach to simulation. As a highly advantageous alternative, we describe herein an automated multiparameter data optimization analysis strategy where the simulated and experimental faradaic current data are compared for both the real and imaginary components in each of the 4th to 12th harmonics after quantifying the charging current data using the time-domain response.
RESUMO
[NiFe]-hydrogenases (Hyd) bind a nickel-iron-based cofactor. The Fe ion of the cofactor is bound by two cyanide ligands and a single carbon monoxide ligand. Minimally six accessory proteins (HypA-HypF) are necessary for NiFe(CN)2CO cofactor biosynthesis in Escherichia coli. It has been shown that the anaerobically purified HypC-HypD-HypE scaffold complex carries the Fe(CN)2CO moiety of this cofactor. In the present study, we have purified the HybG-HypDE complex and used it to successfully reconstitute in vitro active Hyd from E. coli. HybG is a homologue of HypC that is specifically required for the maturation of Hyd-2 and also functions in the maturation of Hyd-1 of E. coli. Maturation of active Hyd-1 and Hyd-2 could be demonstrated in extracts derived from HybG- and HypD-deficient E. coli strains by adding anaerobically purified HybG-HypDE complex. In vitro maturation was dependent on ATP, carbamoylphosphate, nickel and reducing conditions. Hydrogenase maturation was prevented when the purified HybG-HypDE complex used in the maturation assay lacked a bound Fe(CN)2CO moiety. These findings demonstrate that it is possible to isolate incompletely processed intermediates on the maturation pathway and to use these to activate apo-forms of [NiFe]-hydrogenase large subunits.
Assuntos
Monóxido de Carbono/química , Endopeptidases/metabolismo , Escherichia coli/enzimologia , Hidrogenase/metabolismo , Monóxido de Carbono/metabolismo , Coenzimas/biossíntese , Coenzimas/química , Cianetos/química , Cianetos/metabolismo , Endopeptidases/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Hidrogenase/química , Ferro/química , Ligantes , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Níquel/químicaRESUMO
[NiFe]-hydrogenases bind a NiFe-(CN)2CO cofactor in their catalytic large subunit. The iron-sulfur protein HypD and the small accessory protein HypC play a central role in the generation of the CO and CN(-) ligands. Infrared spectroscopy identified signatures on an anaerobically isolated HypCD complex that are reminiscent of those in the hydrogenase active site, suggesting that this complex is the assembly site of the Fe-(CN)2CO moiety of the cofactor prior to its transfer to the hydrogenase large subunit. Here, we report that HypD isolated in the absence of HypC shows infrared bands at 1956 cm(-1), 2072 cm(-1), and 2092 cm(-1) that can be assigned to CO, CN(1), and CN(2), respectively, and which are indistinguishable from those observed for the HypCD complex. HypC could not be isolated with CO or CN(-) ligand contribution. Treatment of HypD with EDTA led to the concomitant loss of Fe and the CO and CN(-) signatures, while oxidation by H2O2 resulted in a positive shift of the CO and CN(-) bands by 35 cm(-1) and 20 cm(-1), respectively, indicative of the ferrous iron as an immediate ligation site for the diatomic ligands. Analysis of HypD amino acid variants identified cysteines 41, 69, and 72 to be essential for maturation of the cofactor. We propose a refined model for the ligation of Fe-(CN)2CO to HypD and the role of HypC in [NiFe]-hydrogenase maturation.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Hidrogenase/química , Hidrogenase/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Domínio Catalítico , Coenzimas/química , Coenzimas/metabolismo , Cianetos/química , Cianetos/metabolismo , Proteínas de Escherichia coli/genética , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Ligantes , Oxirredução , Subunidades Proteicas , Proteínas/genética , Espectroscopia de Infravermelho com Transformada de Fourier , Thermococcus/enzimologiaRESUMO
Endoprotease-specific C-terminal processing is required to complete the maturation of the large subunit of [NiFe]-hydrogenases. This happens only after synthesis and insertion of the NiFe(CN)(2)CO cofactor by the Hyp maturases has occurred. It is assumed that in the absence of maturation the unprocessed species of the large subunit lacks cofactors. In this study we isolated a variant of the hydrogenase 2 large subunit, HybC, containing a fused C-terminal pentapeptide. The polypeptide could not be processed and was unable to associate with the small subunit to deliver an active enzyme. The His(6)-HybC variant protein isolated was brown and had sub-stoichiometric amounts of an oxygen-sensitive Iron-sulfur cluster, which could be chemically reconstituted to a [4Fe-4S] cluster. This cluster was coordinated by the conserved cysteinyl residues that normally ligate the NiFe(CN)(2)CO cofactor. Our findings provide evidence for temporary promiscuity of cofactor-binding sites.
Assuntos
Escherichia coli/enzimologia , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Oxirredutases/química , Oxigênio/química , Carboxil e Carbamoil Transferases/genética , Proteínas de Transporte/genética , Proteínas de Escherichia coli/genética , Hidrogenase/genética , Proteínas Ferro-Enxofre/genética , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Oxirredutases/genéticaRESUMO
BACKGROUND: Escherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H2: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity. RESULTS: Here we report the identification of a new H2: benzyl viologen oxidoreductase enzyme activity in E. coli that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of E. coli mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes. CONCLUSIONS: The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of Escherichia coli have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme.
Assuntos
Benzil Viologênio/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Formiato Desidrogenases/metabolismo , Hidrogênio/metabolismo , Oxirredutases/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Escherichia coli/metabolismo , Espectrometria de Massas , Oxirredução , Selenoproteínas/metabolismoRESUMO
Escherichia coli can both oxidize hydrogen and reduce protons. These activities involve three distinct [NiFe]-hydrogenases, termed Hyd-1, Hyd-2, and Hyd-3, each minimally comprising heterodimers of a large subunit, containing the [NiFe] active site, and a small subunit, bearing iron-sulfur clusters. Dihydrogen-oxidizing activity can be determined using redox dyes like benzyl viologen (BV); however, it is unclear whether electron transfer to BV occurs directly at the active site, or via an iron-sulfur center in the small subunit. Plasmids encoding Strep-tagged derivatives of the large subunits of the three E. coli [NiFe]-hydrogenases restored activity of the respective hydrogenase to strain FTD147, which carries in-frame deletions in the hyaB, hybC, and hycE genes encoding the large subunits of Hyd-1, Hyd-2, and Hyd-3, respectively. Purified Strep-HyaB was associated with the Hyd-1 small subunit (HyaA), and purified Strep-HybC was associated with the Hyd-2 small subunit (HybO), and a second iron-sulfur protein, HybA. However, Strep-HybC isolated from a hybO mutant had no other associated subunits and lacked BV-dependent hydrogenase activity. Mutants deleted separately for hyaA, hybO, or hycG (Hyd-3 small subunit) lacked BV-linked hydrogenase activity, despite the Hyd-1 and Hyd-2 large subunits being processed. These findings demonstrate that hydrogenase-dependent reduction of BV requires the small subunit.
Assuntos
Benzil Viologênio/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Domínio Catalítico , Transporte de Elétrons , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Hidrogenase/genética , Proteínas Ferro-Enxofre/genética , Mutação , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , PlasmídeosRESUMO
[NiFe]-hydrogenases catalyze uptake and evolution of H2 in a wide range of microorganisms. The enzyme is characterized by an inorganic nickel/ iron cofactor, the latter of which carries carbon monoxide and cyanide ligands. In vivo generation of these ligands requires a number of auxiliary proteins, the so-called Hyp family. Initially, HypF binds and activates the precursor metabolite carbamoyl phosphate. HypF catalyzes removal of phosphate and transfers the carbamate group to HypE. In an ATP-dependent condensation reaction, the C-terminal cysteinyl residue of HypE is modified to what has been interpreted as thiocyanate. This group is the direct precursor of the cyanide ligands of the [NiFe]-hydrogenase active site cofactor. We present a FT-IR analysis of HypE and HypF as isolated from E. coli. We follow the HypF-catalyzed cyanation of HypE in vitro and screen for the influence of carbamoyl phosphate and ATP. To elucidate on the differences between HypE and the HypEF complex, spectro-electrochemistry was used to map the vibrational Stark effect of naturally cyanated HypE. The IR signature of HypE could ultimately be assigned to isothiocyanate (-N=C=S) rather than thiocyanate (-S-C≡N). This has important implications for cyanyl-group channeling during [NiFe]-hydrogenase cofactor generation.
Assuntos
Proteínas de Bactérias/metabolismo , Isotiocianatos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Isotiocianatos/química , Dados de Sequência Molecular , Ligação Proteica , Ralstonia/enzimologiaRESUMO
The class of [NiFe]-hydrogenases is characterized by a bimetallic cofactor comprising low-spin nickel and iron ions, the latter of which is modified with a single carbon monoxide (CO) and two cyanide (CN-) molecules. Generation of these ligands in vivo requires a complex maturation apparatus in which the HypC-HypD complex acts as a 'construction site' for the Fe-(CN)2CO portion of the cofactor. The order of addition of the CO and CN- ligands determines the ultimate structure and catalytic efficiency of the cofactor; however much debate surrounds the succession of events. Here, we present an FT-IR spectroscopic analysis of HypC-HypD isolated from a hydrogenase-competent wild-type strain of Escherichia coli. In contrast to previously reported samples, HypC-HypD showed spectral contributions indicative of an electron-rich Fe-CO cofactor, at the same time lacking any Fe-CN- signatures. This immature iron site binds external CO and undergoes oxidative damage when in contact with O2. Binding of CO protects the site against loss of spectral features associated with O2 damage. Our findings strongly suggest that CO ligation precedes cyanation in vivo. Furthermore, the results provide a rationale for the deleterious effects of O2 on in vivo cofactor biosynthesis.
Assuntos
Monóxido de Carbono/química , Coenzimas/química , Cianetos/química , Escherichia coli , Oxigênio/metabolismo , Coenzimas/biossíntese , Proteínas de Escherichia coli/química , Peróxido de Hidrogênio/química , Ferro/química , Cinética , Níquel/química , Proteínas/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Escherichia coli has four hydrogenases (Hyd), three genes of which are encoded by the hya, hyb, and hyc operons. The proton-reducing and hydrogen-oxidizing activities of Hyd-2 (hyb) were analyzed in whole cells grown to stationary phase and cell extracts, respectively, during glycerol fermentation using novel double mutants. H2 production rate at pH 7.5 was decreased by ~3.5- and ~7-fold in hya and hyc (HDK 103) or hyb and hyc (HDK 203) operon double mutants, respectively, compared with the wild type. At pH 6.5, H2 production decreased by ~2- and ~5-fold in HDK103 and HDK203, respectively, compared with the wild type. At pH 5.5, H2 production was reduced by ~4.5-fold in the mutants compared with the wild type. The total hydrogen-oxidizing activity was shown to depend on the pH of the growth medium in agreement with previous findings and was significantly reduced in the HDK103 or HDK203 mutants. At pH 7.5, Hyd-2 activity was 0.26 U (mg protein)(-1) and Hyd-1 activity was 0.1 U (mg protein)(-1). As the pH of the growth medium decreased to 6.5, Hyd-2 activity was 0.16 U (mg protein)(-1), and Hyd-1 was absent. Surprisingly, at pH 5.5, there was an increase in Hyd-2 activity (0.33 U mg protein)(-1) but not in that of Hyd-1. These findings show a major contribution of Hyd-2 to H2 production during glycerol fermentation that resulted from altered metabolism which surprisingly influenced proton reduction.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Fermentação , Glicerol/metabolismo , Hidrogenase/metabolismo , Meios de Cultura/metabolismo , Citoplasma/enzimologia , Ativação Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Hidrogenase/genética , Oxirredução , Deleção de SequênciaRESUMO
[NiFe]-hydrogenase accessory proteins HypC and HypD form a complex that binds a Fe-(CN)2CO moiety and CO2. In this study two HypC homologues from Escherichia coli were purified under strictly anaerobic conditions and both contained sub-stoichiometric amounts of iron (approx. 0.3 molFe/mol HypC). Infrared spectroscopic analysis identified a signature at 2337 cm⻹ indicating bound CO2. Aerobically isolated HypC lacked both Fe and CO2. Exchange of either of the highly conserved amino acid residues Cys2 or His51 abolished both Fe- and CO2-binding. Our results suggest that HypC delivers CO2 bound directly to Fe for reduction to CO by HypD.
Assuntos
Dióxido de Carbono/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Hidrogenase/química , Ferro/química , Chaperonas Moleculares/química , Proteínas de Transporte/química , Cisteína/química , Histidina/química , Peróxido de Hidrogênio/química , Ligantes , Oxirredução , Oxigênio/químicaRESUMO
The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)(2)CO cofactor in the large subunit of [NiFe]-hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep-tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN(-) ligands as well as CO(2). Metal and sulphide analyses revealed that along with the [4Fe-4S](2+) cluster in HypD, the complex has two additional oxygen-labile Fe ions. We prove that HypD cysteine 41 is required for the coordination of all three ligands. These findings suggest that the HypCD complex carries minimally the Fe(CN)(2)CO cofactor.
Assuntos
Monóxido de Carbono/metabolismo , Cianetos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Hidrogenase/metabolismo , Proteínas/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Sítios de Ligação , Monóxido de Carbono/química , Cátions Bivalentes , Coenzimas/química , Coenzimas/metabolismo , Cianetos/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidrogenase/química , Hidrogenase/genética , Ferro/química , Ferro/metabolismo , Ligantes , Plasmídeos , Ligação Proteica , Proteínas/química , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometria InfravermelhoRESUMO
[NiFe]-hydrogenases are multimeric proteins. The large subunit contains the NiFe(CN)(2)CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)(2)CO active center requires six Hyp accessory proteins. The synthesis of the CN(-) ligands is catalyzed by the combined actions of HypF and HypE using carbamoylphosphate as a substrate. We report the structure of Escherichia coli HypF(92-750) lacking the N-terminal acylphosphatase domain. HypF(92-750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn(2+) ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by â¼14 Å. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.
Assuntos
Carboxil e Carbamoil Transferases/química , Proteínas de Escherichia coli/química , Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Sítios de Ligação , Carbamoil-Fosfato/metabolismo , Carboxil e Carbamoil Transferases/metabolismo , Catálise , Domínio Catalítico , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrogenase/química , Ligantes , AcilfosfataseRESUMO
By combining extracts from strains lacking genes encoding either the maturation enzymes or the large subunits of hydrogenases 1, 2 and 3 we could reconstitute in vitro under strictly anaerobic conditions 10-15% of the hydrogenase activity present in wild type Escherichia coli extracts. Purified, unprocessed Strep-tagged variants of the hydrogenase 2 large subunit, HybC, isolated from either a ΔhybD (encoding the hydrogenase 2-specific protease) mutant or a strain deficient in HypF could also be matured to active, processed enzyme using this system. These studies reveal that minimally one step early on the hydrogenase maturation pathway is oxygen-labile.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Hidrogenase/metabolismo , Oxigênio/metabolismo , Carboxil e Carbamoil Transferases/genética , Endopeptidases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidrogenase/química , Hidrogenase/genéticaRESUMO
The in vitro reconstitution of molybdenum nitrogenase was manipulated to generate a chimeric enzyme in which the active site iron-molybdenum cofactor (FeMo-co) is replaced by NifB-co. The NifDK/NifB-co enzyme was unable to reduce N(2) to NH(3), while exhibiting residual C(2)H(4) and considerable H(2) production activities. Production of H(2) by NifDK/NifB-co was stimulated by N(2) and was dependent on NifH and ATP hydrolysis. Thus, NifDK/NifB-co is a useful tool to gain insights into the catalytic mechanism of nitrogenase. Furthermore, phylogenetic analysis of D and K homologs indicates that several early emerging lineages, which contain NifB, NifH and NifDK encoding genes but which lack other genes required for processing NifB-co into FeMo-co, might encode an enzyme with similar catalytic properties to NifDK/NifB-co.
Assuntos
Domínio Catalítico , Evolução Molecular , Compostos de Ferro/metabolismo , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Acetileno/metabolismo , Trifosfato de Adenosina/metabolismo , Hidrogênio/metabolismo , Klebsiella pneumoniae/enzimologia , Modelos Moleculares , Nitrogênio/metabolismo , Oxirredução , Filogenia , Especificidade por SubstratoRESUMO
Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron-molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe(2+), S(2-), MoO4(2-), and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe(2+), S(2-), MoO4(2-), R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed.
Assuntos
Molibdoferredoxina/síntese química , Fixação de Nitrogênio , Nitrogenase/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Indicadores e Reagentes , Ferro , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Molibdênio , Enxofre , Ácidos TricarboxílicosRESUMO
The iron-molybdenum cofactor of nitrogenase (FeMo-co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre-synthesized apo-dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo-co precursors or FeMo-co during cofactor synthesis. In this work, the binding of FeMo-co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo-co synthesis. Purified NifX binds NifB cofactor (NifB-co), a precursor to FeMo-co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe-S] cluster that serves as a FeMo-co precursor, and we have designated it as the VK-cluster. In contrast to NifB-co, the VK-cluster is electronic paramagnetic resonance (EPR)-active in the reduced and the oxidized states. The NifX/VK-cluster complex is unable to support in vitro FeMo-co synthesis in the absence of NifEN because further processing of the VK-cluster into FeMo-co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo-co precursors and thus help control their flux during FeMo-co synthesis.
Assuntos
Azotobacter vinelandii/enzimologia , Proteínas de Bactérias/metabolismo , Compostos de Ferro/metabolismo , Molibdoferredoxina/biossíntese , Fixação de Nitrogênio/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Vias Biossintéticas , Genes Bacterianos , Molibdoferredoxina/químicaRESUMO
The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. A His-tagged version of the NifEN complex has been previously purified and shown to carry two identical [4Fe-4S] clusters of unknown function and a [Fe-S]-containing FeMo-co precursor. We have improved the purification of the his-NifEN protein from a DeltanifHDK strain of Azotobacter vinelandii and have found that the amounts of iron and molybdenum within NifEN were significantly higher than those reported previously. In an in vitro FeMo-co synthesis system with purified components, the NifEN protein served as a source of both molybdenum and a [Fe-S]-containing FeMo-co precursor, showing significant FeMo-co synthesis activity in the absence of externally added molybdate. Thus, the NifEN scaffold protein, purified from DeltanifHDK background, contained the Nif-Bco-derived Fe-S cluster and molybdenum, although these FeMo-co constituents were present at different levels within the protein complex.