Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.12.7.2 (hydrogenase)
3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Analysis of the HypC-hycE complex, a key intermediate in the assembly of the metal center of the Escherichia coli hydrogenase 3. 1078 87

The hydrogenase in Azotobacter vinelandii, like other membrane-bound [NiFe] hydrogenases, consists of a catalytic heterodimer and an integral membrane cytochrome b. The histidines ligating the hemes in this cytochrome b were identified by H(2) oxidation properties of altered proteins produced by site-directed mutagenesis. Four fully conserved and four partially conserved histidines in HoxZ were substituted with alanine or tyrosine. The roles of these histidines in HoxZ heme binding and hydrogenase were characterized by O(2)-dependent H(2) oxidation and H(2)-dependent methylene blue reduction in vivo. Mutants H33A/Y (H33 replaced by A or Y), H74A/Y, H194A, H208A/Y, and H194,208A lost O(2)-dependent H(2) oxidation activity, H194Y and H136A had partial activity, and H97Y,H98A and H191A had full activity. These results suggest that the fully conserved histidines 33, 74, 194, and 208 are ligands to the hemes, tyrosine can serve as an alternate ligand in position 194, and H136 plays a role in H(2) oxidation. In mutant H194A/Y, imidazole (Imd) rescued H(2) oxidation activity in intact cells, which suggests that Imd acts as an exogenous ligand. The heterodimer activity, quantitatively determined as H(2)-dependent methylene blue reduction, indicated that the heterodimers of all mutants were catalytically active. H33A/Y had wild-type levels of methylene blue reduction, but the other HoxZ ligand mutants had significantly less than wild-type levels. Imd reconstituted full methylene blue reduction activity in mutants H194A/Y and H208A/Y and partial activity in H194,208A. These results indicate that structural and functional integrity of HoxZ is required for physiologically relevant H(2) oxidation, and structural integrity of HoxZ is necessary for full heterodimer-catalyzed H(2) oxidation.
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PMID:The hydrogenase cytochrome b heme ligands of Azotobacter vinelandii are required for full H(2) oxidation capability. 1085 74

Lysines 9 and 10 in Desulfuromonas acetoxidans cytochrome c7, which could be involved in the interaction mechanism with the redox partners, have been replaced by alanine residues using site-directed mutagenesis. The solution structure of the fully oxidized form of K9-10A cytochrome c7, which is paramagnetic with three paramagnetic centers, has been determined via 1H NMR. The assignment of the spectra has been performed through an automatic program whose algorithm and strategy are here described. The assignment of the NOESY spectra has been further extended by back calculating the NOESY maps. The final number of meaningful NOE-based upper distance limits was 1186. In the Restrained Energy Minimization calculations, 147 pseudocontact shift constraints were also included, which showed consistency with NOE-based constraints and therefore further contribute to validate the structure quality. A final family of 35 conformers was calculated with RMSD values with respect to the mean structure of 0.69 +/- 0.17 A and 1.05 +/- 0.14 A for the backbone and heavy atoms, respectively. The overall fold of the molecule is maintained with respect to the native protein. The loop present between heme III and heme IV results to be highly disordered also in the present structure although its overall shape mainly resembles that of the oxidized native protein, and the two strands which give rise to the short beta-sheet present at the N-terminus and connected by a turn containing the mutated residues, are less clearly defined. If this loop is neglected, the RMSD values are 0.52 +/- 0.07 A and 0.92 +/- 0.06 A for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The relative distances and orientations of the three hemes are maintained, as well as the orientation of the imidazole rings of the axial histidine ligands, with the only exception of heme IV. Such difference probably reflects minor conformational changes due to the substitution of the vicinal Lys 10 with an Ala. The replacement of the two lysines does not affect the reduction potentials of the three hemes, consistently with the expectations on the basis of the structure and electrostatic calculations. However, the replacement of the two lysines affects the reactivity of the mutant cytochrome c7 with [Fe] hydrogenase, inducing a change in Km. This finding is in agreement with the identification of the protein area around heme IV as the interacting site.
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PMID:A quick solution structure determination of the fully oxidized double mutant K9-10A cytochrome c7 from Desulfuromonas acetoxidans and mechanistic implications. 1188 73

Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys(531) exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His(265) exhibits hydroxylamine reductase activity. Both Cys(531) and His(265) are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.
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PMID:Converting the NiFeS carbon monoxide dehydrogenase to a hydrogenase and a hydroxylamine reductase. 1237 22

A novel extremely haloalkaliphilic, strictly anaerobic, acetogenic bacterium strain APO was isolated from sediments of the athalassic, meromictic, alkaline Mono Lake in California. The Gram-positive, spore-forming, slightly curved rods with sizes 0.55-0.7x1.7-3.0 microm were motile by a single laterally attached flagellum. Strain APO was mesophilic (range 10-48 degrees C, optimum of 37 degrees C); halophilic (NaCl range 1-20% (w/v) with optimum of 3-5% (w/v), and alkaliphilic (pH range 8.0-10.5, optimum 9.5). The novel isolate required sodium ions in the medium. Strain APO was an organotroph with a fermentative type of metabolism and used the substrates peptone, bacto-tryptone, casamino acid, yeast extract, l-serine, l-lysine, l-histidine, l-arginine, and pyruvate. The new isolate performed the Stickland reaction with the following amino acid pairs: proline + alanine, glycine + alanine, and tryptophan + valine. The main end product of growth was acetate. High activity of CO dehydrogenase and hydrogenase indicated the presence of a homoacetogenic, non-cycling acetyl-CoA pathway. Strain APO was resistant to kanamycin but sensitive to chloramphenicol, tetracycline, and gentamycin. The G+C content of the genomic DNA was 44.4 mol% (by HPLC method). The sequence of the 16S rRNA gene of strain APO possessed 98.2% similarity with the sequence from Tindallia magadiensis Z-7934, but the DNA-DNA hybridization value between these organisms was only 55%. On the basis of these physiological and molecular properties, strain APO is proposed to be a novel species of the genus Tindallia with the name Tindallia californiensis sp. nov., (type strain APO = ATCC BAA-393 = DSM 14871).
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PMID:Tindallia californiensis sp. nov., a new anaerobic, haloalkaliphilic, spore-forming acetogen isolated from Mono Lake in California. 1272 59

The number of accessible SH-groups was determined in membrane vesicles prepared from Escherichia coli growing in fermentation conditions at slightly alkaline pH on glucose with or without added formate. Addition of ATP or formate to the vesicles caused a approximately 1.4-fold increase in the number of accessible SH-groups. The increase was inhibited by treatment with N-ethylmaleimide or the presence of the F(0)F(1)-ATPase inhibitors N,N(')-dicyclohexylcarbodiimide or sodium azide. The increase in accessible SH-groups was also absent in strains with the ATP synthase operon deleted or with the single F(0) domain cysteine Cysb21 changed to Ala. Using hyc and hyf mutants, it was shown that the increase was also largely dependent on hydrogenase 4 or hydrogenase 3, main components of formate hydrogen lyase, when bacteria were grown in the absence or presence of added formate. These results suggest a relationship between the F(0)F(1)-ATP synthase and hydrogenase 4 or hydrogenase 3 under fermentation conditions.
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PMID:The number of accessible SH-groups in Escherichia coli membrane vesicles is increased by ATP or by formate. 1291

The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.
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PMID:Characterization of the menaquinone reduction site in the diheme cytochrome b membrane anchor of Wolinella succinogenes NiFe-hydrogenase. 1457 51

The tetraheme cytochrome c3 isolated from Desulfomicrobium baculatum (DSM 1743)(Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c3 isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 --> Ser, Val-45 --> Ala, and Phe-88 --> Tyr. X-ray crystallography was used to determine the structure of cytochrome c3 from Dsmb, showing that it is very similar to the published structure of cytochrome c3 from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c3 was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c3. This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats.
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PMID:Proton-assisted two-electron transfer in natural variants of tetraheme cytochromes from Desulfomicrobium Sp. 1545 79

Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H(2)), but little is known about the diffusion of H(2) toward the active site. Here we analyze pathways for H(2) permeation using molecular dynamics (MD) simulations in explicit solvent. Various MD simulation replicates were done, to improve the sampling of the system states. H(2) easily permeates hydrogenase in every simulation and it moves preferentially in channels. All H(2) molecules that reach the active site made their approach from the side of the Ni ion. H(2) is able to reach distances of <4 A from the active site, although after 6 A permeation is difficult. In this region we mutated Val-67 into alanine and perform new MD simulations. These simulations show an increase of H(2) inside the protein and at lower distances from the active site. This valine can be a control point in the H(2) access to the active center.
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PMID:Pathways of H2 toward the active site of [NiFe]-hydrogenase. 1673 62

The increasing importance of hydrogenase enzymes in the new energy research field has led us to examine the structure and dynamics of potential hydrogenase mimics, based on a ferrocene-peptide scaffold, using molecular dynamics (MD) simulations. To enable this MD study, a molecular mechanics force field for ferrocene-bearing peptides was developed and implemented in the CHARMM simulation package, thus extending the usefulness of the package into peptide-bioorganometallic chemistry. Using the automated frequency-matching method (AFMM), optimized intramolecular force-field parameters were generated through quantum chemical reference normal modes. The partial charges for ferrocene were derived by fitting point charges to quantum-chemically computed electrostatic potentials. The force field was tested against experimental X-ray crystal structures of dipeptide derivatives of ferrocene-1,1'-dicarboxylic acid. The calculations reproduce accurately the molecular geometries, including the characteristic C2-symmetrical intramolecular hydrogen-bonding pattern, that were stable over 0.1 micros MD simulations. The crystal packing properties of ferrocene-1-(D)alanine-(D)proline-1'-(D)alanine-(D)proline were also accurately reproduced. The lattice parameters of this crystal were conserved during a 0.1 micros MD simulation and match the experimental values almost exactly. Simulations of the peptides in dichloromethane are also in good agreement with experimental NMR and circular dichroism (CD) data in solution. The developed force field was used to perform MD simulations on novel, as yet unsynthesized peptide fragments that surround the active site of [Ni-Fe] hydrogenase. The results of this simulation lead us to propose an improved design for synthetic peptide-based hydrogenase models. The presented MD simulation results of metallocenes thereby provide a convincing validation of our proposal to use ferrocene-peptides as minimal enzyme mimics.
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PMID:Force-field development and molecular dynamics simulations of ferrocene-peptide conjugates as a scaffold for hydrogenase mimics. 1776 6


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