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1.
Anal Chem ; 88(14): 7060-7, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27328020

RESUMO

Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment.


Assuntos
Gases/efeitos da radiação , Espectrometria de Massas/métodos , Proteínas de Membrana/efeitos da radiação , Complexos Multiproteicos/efeitos da radiação , Acidianus/enzimologia , Avidina/química , Avidina/efeitos da radiação , Chaperonina 60/química , Chaperonina 60/efeitos da radiação , Gases/química , Raios Infravermelhos , Proteínas de Membrana/química , Micelas , Complexos Multiproteicos/química , Fosfatidilgliceróis/química , Subunidades Proteicas/química , Subunidades Proteicas/efeitos da radiação , Thermus thermophilus/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/efeitos da radiação
2.
Wei Sheng Wu Xue Bao ; 54(10): 1193-203, 2014 Oct 04.
Artigo em Chinês | MEDLINE | ID: mdl-25803897

RESUMO

OBJECTIVE: To study the adaptation of A. hospitalis W1 to oligotrophic and acidic hot spring environments at the whole genome level. METHODS: We annotated the gene functions and constructed metabolic pathways of strain W1 by using different databases, such as NCBI non-redundant database (NRDB), UniProt, Sulfolobus protein database and Kyoto Encyclopedia of Genes and Genomes (KEGG). The metabolic pathways were polished according to the results of comparative genomics. RESULTS: Strain W1 grew autotrophically by fixing CO2 as carbon source through 3-hydroxypropionate/4-hydroxybutyrate or dicarboxylate-4-hydroxybutyrate cycle, and gained energy for growth by oxidation of reduced inorganic sulfur compounds (RISCs). Strain W1 differenced from A. ambivalens because its genome did not possess sulfur-metabolizing genes encoding sulfite: acceptor oxidoreductase, adenosine phosphosulfate reductase, sulfate adenylyl transferase and phosphoadenosine phosphosulfate reductase. Glucose was metabolized by strain W1 through non- phosphorylated Entner-Doudoroff pathway and tricarboxylic acid cycle. In addition, the sugar and amino acids transporters, as well as related hydrolysis enzymes were identified in the genome. These results suggest that strain W1 could also grow facultative autotrophically. Strain W1 cannot use H2 as electron donor due to lack of hydrogenase encoding genes. CONCLUSION: The versatile metabolic patterns afforded A. hospitalis W1 the ability to adapt to oligotrophic and acidic hot spring environments. Furthermore, the unique metabolic features of strain W1 will help to better understand the metabolic diversities of Acidianus.


Assuntos
Acidianus/fisiologia , Fontes Termais/microbiologia , Acidianus/enzimologia , Acidianus/genética , Acidianus/isolamento & purificação , Ácidos/metabolismo , Adaptação Fisiológica , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Processos Autotróficos , Ciclo do Ácido Cítrico , Genoma Arqueal , Fontes Termais/química , Redes e Vias Metabólicas , Enxofre/metabolismo
3.
J Bacteriol ; 195(18): 4046-56, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23836868

RESUMO

Carbon disulfide (CS(2)) and carbonyl sulfide (COS) are important in the global sulfur cycle, and CS(2) is used as a solvent in the viscose industry. These compounds can be converted by sulfur-oxidizing bacteria, such as Acidithiobacillus thiooxidans species, to carbon dioxide (CO(2)) and hydrogen sulfide (H2S), a property used in industrial biofiltration of CS(2)-polluted airstreams. We report on the mechanism of bacterial CS(2) conversion in the extremely acidophilic A. thiooxidans strains S1p and G8. The bacterial CS(2) hydrolases were highly abundant. They were purified and found to be homologous to the only other described (archaeal) CS(2) hydrolase from Acidianus strain A1-3, which forms a catenane of two interlocked rings. The enzymes cluster in a group of ß-carbonic anhydrase (ß-CA) homologues that may comprise a subclass of CS(2) hydrolases within the ß-CA family. Unlike CAs, the CS(2) hydrolases did not hydrate CO(2) but converted CS(2) and COS with H(2)O to H(2)S and CO(2). The CS(2) hydrolases of A. thiooxidans strains G8, 2Bp, Sts 4-3, and BBW1, like the CS(2) hydrolase of Acidianus strain A1-3, exist as both octamers and hexadecamers in solution. The CS(2) hydrolase of A. thiooxidans strain S1p forms only octamers. Structure models of the A. thiooxidans CS(2) hydrolases based on the structure of Acidianus strain A1-3 CS(2) hydrolase suggest that the A. thiooxidans strain G8 CS(2) hydrolase may also form a catenane. In the A. thiooxidans strain S1p enzyme, two insertions (positions 26 and 27 [PD] and positions 56 to 61 [TPAGGG]) and a nine-amino-acid-longer C-terminal tail may prevent catenane formation.


Assuntos
Acidianus/enzimologia , Acidithiobacillus thiooxidans/enzimologia , Proteínas Arqueais/química , Proteínas de Bactérias/química , Dissulfeto de Carbono/metabolismo , Hidrolases/química , Homologia de Sequência de Aminoácidos , Acidianus/genética , Acidithiobacillus thiooxidans/genética , Sequência de Aminoácidos , Antracenos/química , Antracenos/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Dissulfeto de Carbono/química , DNA Bacteriano/análise , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato
4.
Chem Commun (Camb) ; 49(71): 7770-2, 2013 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-23771150

RESUMO

CS2 hydrolase, a zinc-dependent enzyme that converts carbon disulfide to carbon dioxide and hydrogen sulfide, exists as a mixture of octameric ring and hexadecameric catenane forms in solution. A combination of size exclusion chromatography, multi-angle laser light scattering, and mass spectrometric analyses revealed that the unusual catenane structure is not an artefact, but a naturally occurring structure.


Assuntos
Antracenos/metabolismo , Hidrolases/metabolismo , Acidianus/enzimologia , Antracenos/química , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Dissulfeto de Carbono/química , Dissulfeto de Carbono/metabolismo , Hidrolases/química , Luz , Estrutura Quaternária de Proteína , Espalhamento de Radiação
5.
Appl Environ Microbiol ; 78(15): 5463-5, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22636008

RESUMO

In the course of screening for virus-host systems in extreme thermal environments, we have isolated a strain of the hyperthermophilic archaeaon Acidianus hospitalis producing unusual filamentous particles with a zipper-like appearance. The particles were shown to represent a secreted form of a genuine cellular enzyme, tetrathionate hydrolase, involved in sulfur metabolism.


Assuntos
Acidianus/enzimologia , Hidrolases/química , Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , Dimerização , Eletroforese em Gel de Poliacrilamida , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Microscopia Eletrônica , Mitomicina , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/ultraestrutura
6.
Nature ; 478(7369): 412-6, 2011 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-22012399

RESUMO

Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H(2)S) and carbon disulphide (CS(2)). The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS(2) into H(2)S and carbon dioxide (CO(2)), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS(2) hydrolase from Acidianus A1-3. The enzyme monomer displays a typical ß-carbonic anhydrase fold and active site, yet CO(2) is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical ß-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS(2), which is hydrophobic. The transposon sequences that surround the gene encoding this CS(2) hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient ß-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS(2) metabolism.


Assuntos
Acidianus/enzimologia , Dissulfeto de Carbono/metabolismo , Evolução Molecular , Hidrolases/genética , Acidianus/classificação , Acidianus/genética , Domínio Catalítico , Cristalografia por Raios X , Hidrolases/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Filogenia , Estrutura Terciária de Proteína
7.
Biochemistry ; 48(24): 5613-22, 2009 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-19438211

RESUMO

A sulfide:quinone oxidoreductase (SQR) was isolated from the membranes of the hyperthermoacidophilic archaeon Acidianus ambivalens, and its X-ray structure, the first reported for an SQR, was determined to 2.6 A resolution. This enzyme was functionally and structurally characterized and was shown to have two redox active sites: a covalently bound FAD and an adjacent pair of cysteine residues. Most interestingly, the X-ray structure revealed the presence of a chain of three sulfur atoms bridging those two cysteine residues. The possible implications of this observation in the catalytic mechanism for sulfide oxidation are discussed, and the role of SQR in the sulfur dependent bioenergetics of A. ambivalens, linked to oxygen reduction, is addressed.


Assuntos
Proteínas Arqueais/química , Quinona Redutases/química , Acidianus/enzimologia , Acidianus/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Peso Molecular , Oxirredução , Quinona Redutases/metabolismo , Relação Estrutura-Atividade , Sulfetos/química , Sulfetos/metabolismo
8.
Biochem Biophys Res Commun ; 369(3): 919-23, 2008 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-18329378

RESUMO

Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.


Assuntos
Acidianus/enzimologia , Proteínas Arqueais/química , Oxirredutases/química , Sítios de Ligação , Catálise , Cristalografia , Conformação Proteica
9.
FEMS Microbiol Lett ; 281(2): 147-54, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18312361

RESUMO

A dihydrolipoamide dehydrogenase (DLDH) was purified and characterized for the first time from a crenarchaeon, Acidianus ambivalens. The holoenzyme consists of two identical subunits with a molecular mass of 45.4 kDa per monomer. It contains FAD as a prosthetic group and uses NAD+ as the preferential substrate, but can also reduce NADP+. The Michaelis-Menten constants of the forward (NAD+ reduction) and reverse (NADH oxidation) reactions were KM (dihydrolipoamide)=0.70 mM, KM (NAD+)=0.71 mM, KM (lipoamide)=1.26 mM and KM (NADH)=3.15 microM. A comparative study of NADH:lipoamide oxidoreductase and NADH:K3[Fe(CN)6] oxidoreductase activities was performed, the optimal temperature and pH being different for each: 55 degrees C, pH 7 and 89 degrees C, pH 5.5, respectively. Although DLDH is generally part of the alpha-ketoacid dehydrogenase complexes in Bacteria and Eukarya, none of these complexes has yet been isolated from Sulfolobales. The metabolic role of DLDH in these organisms is discussed.


Assuntos
Acidianus/enzimologia , Proteínas Arqueais/química , Di-Hidrolipoamida Desidrogenase/química , Acidianus/genética , Proteínas Arqueais/isolamento & purificação , Di-Hidrolipoamida Desidrogenase/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de Proteína , Análise Espectral , Temperatura
10.
Science ; 311(5763): 996-1000, 2006 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-16484493

RESUMO

Numerous microorganisms oxidize sulfur for energy conservation and contribute to the global biogeochemical sulfur cycle. We have determined the 1.7 angstrom-resolution structure of the sulfur oxygenase reductase from the thermoacidophilic archaeon Acidianus ambivalens, which catalyzes an oxygen-dependent disproportionation of elemental sulfur. Twenty-four monomers form a large hollow sphere enclosing a positively charged nanocompartment. Apolar channels provide access for linear sulfur species. A cysteine persulfide and a low-potential mononuclear non-heme iron site ligated by a 2-His-1-carboxylate facial triad in a pocket of each subunit constitute the active sites, accessible from the inside of the sphere. The iron is likely the site of both sulfur oxidation and sulfur reduction.


Assuntos
Acidianus/enzimologia , Proteínas Arqueais/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Enxofre/metabolismo , Acidianus/fisiologia , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Ferro/química , Ferro/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Eletricidade Estática
11.
Biochim Biophys Acta ; 1764(4): 842-5, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16303338

RESUMO

NADH:quinone oxidoreductases (NDHs), constitute one of the electron entry points into membrane-bound respiratory chains, oxidising NADH and reducing quinones. Type-II NDHs (NDH-2) are functionally unable to translocate protons and are typically constituted by a single approximately 50 kDa subunit lacking iron-sulfur clusters and containing one flavin as the sole redox centre. No three dimensional crystal structure is yet available for NDHs. We describe the crystallisation and preliminary structure determination of a NDH-2 that contains a covalently bound FAD, isolated from the membrane fraction of Acidianus ambivalens, a hyperthermoacidophilic archaeon capable of growing at 80 degrees C and pH 2.0. NDH-2 was solubilised with the detergent n-dodecyl-beta-d-maltoside and crystallised using ammonium phosphate as precipitant. The structure was solved by MIRAS using Pt and I derivatives.


Assuntos
Acidianus/enzimologia , Quinona Redutases/química , Cristalização , Cristalografia por Raios X
12.
J Biol Inorg Chem ; 10(6): 625-35, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16163550

RESUMO

The coordination environment of the Cu(B) center of the quinol oxidase from Acidianus ambivalens, a type B heme-copper oxygen reductase, was investigated by Fourier transform (FT) IR and extended X-ray absorption fine structure (EXAFS) spectroscopy. The comparative structural chemistry of dinuclear Fe-Cu sites of the different types of oxygen reductases is of great interest. Fully reduced A. ambivalens quinol oxidase binds CO at the heme a (3) center, with nu(CO)=1,973 cm(-1). On photolysis, the CO migrated to the Cu(B) center, forming a Cu (B) (I) -CO complex with nu(CO)=2,047 cm(-1). Raising the temperature of the samples to 25 degrees C did not result in a total loss of signal in the FTIR difference spectrum although the intensity of these signals was reduced sevenfold. This observation is consistent with a large energy barrier against the geminate rebinding of CO to the heme iron from Cu(B), a restricted limited access at the active-site pocket for a second binding, and a kinetically stable Cu(B)-CO complex in A. ambivalens aa (3). The Cu(B) center was probed in a number of different states using EXAFS spectroscopy. The oxidized state was best simulated by three histidines and a solvent O scatterer. On reduction, the site became three-coordinate, but in contrast to the bo (3) enzyme, there was no evidence for heterogeneity of binding of the coordinated histidines. The Cu(B) centers in both the oxidized and the reduced enzymes also appeared to contain substoichiometric amounts (0.2 mol equiv) of nonlabile chloride ion. EXAFS data of the reduced carbonylated enzyme showed no difference between dark and photolyzed forms. The spectra could be well fit by 2.5 imidazoles, 0.5 Cl(-) and 0.5 CO ligands. This arrangement of scatterers would be consistent with about half the sites remaining as unligated Cu(his)(3) and half being converted to Cu(his)(2)Cl(-)CO, a 50/50 ratio of Cu(his)(2)Cl(-) and Cu(his)(3)CO, or some combination of these formulations.


Assuntos
Acidianus/enzimologia , Cobre/química , Heme/química , Oxirredutases/química , Estrutura Molecular , Oxirredução , Espectroscopia de Infravermelho com Transformada de Fourier
13.
FEMS Microbiol Lett ; 248(2): 171-6, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15970399

RESUMO

The sulfur oxygenase reductase (SOR) is the initial enzyme in the sulfur oxidation pathway of Acidianus ambivalens. The SOR is composed of 308 aa residues, three of which are cysteines, and contains a mononuclear non-heme iron site. Mutations of the suspected iron-binding residues H86, H90 and E114 to alanine resulted in inactive enzyme with no iron incorporated, whereas an E114D mutant showed 1% of wild type activity. The mutation of C31 to alanine and serine caused inactivity of the enzyme, however, the iron content was the same as in the wild type. C101A, C104S/A, and C101/104S/A double mutants caused a decrease in specific activity to 10-43% of the wild type while the C101S mutant showed only 1% activity of the wild type. The drop in activity of the C101S and E114D mutants was accompanied with a proportional decrease in iron content. In all cases the oxygenase and reductase partial reactions were equally affected. It was concluded that the Fe site with H86, H90 and E114 as ligands and C31 constitute the core active site whereas C101 and C104 optimize reaction conditions.


Assuntos
Acidianus/enzimologia , Proteínas Arqueais/química , Oxirredutases/química , Acidianus/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação/genética , Cisteína , Ferro , Mutagênese , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre
14.
Biochim Biophys Acta ; 1747(2): 267-70, 2005 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-15698962

RESUMO

The sulfur oxygenase reductase (SOR) is the initial enzyme in the aerobic sulfur metabolism of the thermoacidophilic and chemolithoautotrophic crenarchaeote Acidianus ambivalens. Single colorless polyhedral crystals were obtained under two crystallization conditions from SOR preparations heterologously overproduced in Escherichia coli. They belonged to space-group I4 and diffraction data were collected up to 1.7 A resolution. Their Patterson symmetry shows additional 4-, 3- and 2-fold non-crystallographic symmetry rotation axes, characteristic of the point group 432. Taking into account the molecular mass of SOR, the crystal unit cell volume, the non-crystallographic symmetry operators and previous electron microscopy studies of the SOR, it was deduced that the quaternary structure of the functionally active enzyme is an icosatetramer with 871 kDa molecular mass.


Assuntos
Acidianus/enzimologia , Proteínas Arqueais/química , Oxirredutases/química , Acidianus/genética , Antígenos de Diferenciação de Linfócitos T/química , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Tamanho Celular , Cristalização , Escherichia coli/genética , Heme/química , Heme/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis , Peso Molecular , Oxirredutases/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Enxofre/química , Enxofre/metabolismo
15.
Appl Environ Microbiol ; 71(2): 621-8, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15691910

RESUMO

Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C(31) and C(101)-X-X-C(104); numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn(2+) strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C(31) and C(101)-X-X-C(104), in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.


Assuntos
Acidianus/enzimologia , Proteínas Arqueais/metabolismo , Cisteína , Regulação da Expressão Gênica em Archaea , Oxirredutases/metabolismo , Frações Subcelulares/enzimologia , Acidianus/genética , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Catálise , Dicroísmo Circular , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Oxirredutases/química , Oxirredutases/genética , Alinhamento de Sequência , Espectrometria de Fluorescência , Enxofre/metabolismo
16.
Biophys J ; 87(6): 4316-25, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15377522

RESUMO

Heme-copper oxidases are membrane proteins found in the respiratory chain of aerobic organisms. They are the terminal electron acceptors coupling the translocation of protons across the membrane with the reduction of oxygen to water. Because the catalytic process occurs in the heme cofactors positioned well inside the protein matrix, proton channels must exist. However, due to the high structural divergence among this kind of proteins, the proton channels previously described are not necessarily conserved. In this work we modeled the structure of the quinol oxidase from Acidianus ambivalens using comparative modeling techniques for identifying proton channels. Additionally, given the high importance that water molecules may have in this process, we have developed a methodology, within the context of comparative modeling, to identify high water probability zones and to deconvolute them into chains of ordered water molecules. From our results, and from the existent information from other proteins from the same superfamily, we were able to suggest three possible proton channels: one K-, one D-, and one Q-spatial homologous proton channels. This methodology can be applied to other systems where water molecules are important for their biological function.


Assuntos
Acidianus/enzimologia , Canais Iônicos/química , Modelos Químicos , Modelos Moleculares , Oxirredutases/química , Bombas de Próton/química , Água/química , Modelos Estatísticos , Oxirredução , Prótons , Relação Estrutura-Atividade
17.
Mol Microbiol ; 53(4): 1147-60, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15306018

RESUMO

Thiosulphate is one of the products of the initial step of the elemental sulphur oxidation pathway in the thermoacidophilic archaeon Acidianus ambivalens. A novel thiosulphate:quinone oxidoreductase (TQO) activity was found in the membrane extracts of aerobically grown cells of this organism. The enzyme was purified 21-fold from the solubilized membrane fraction. The TQO oxidized thiosulphate with tetrathionate as product and ferricyanide or decyl ubiquinone (DQ) as electron acceptors. The maximum specific activity with ferricyanide was 73.4 U (mg protein)(-1) at 92 degrees C and pH 6, with DQ it was 397 mU (mg protein)(-1) at 80 degrees C. The Km values were 2.6 mM for thiosulphate (k(cat) = 167 s(-1)), 3.4 mM for ferricyanide and 5.87 micro M for DQ. The enzymic activity was inhibited by sulphite (Ki = 5 micro M), metabisulphite, dithionite and TritonX-100, but not by sulphate or tetrathionate. A mixture of caldariella quinone, sulfolobus quinone and menaquinone was non-covalently bound to the protein. No other cofactors were detected. Oxygen consumption was measured in membrane fractions upon thiosulphate addition, thus linking thiosulphate oxidation to dioxygen reduction, in what constitutes a novel activity among Archaea. The holoenzyme was composed of two subunits of apparent molecular masses of 28 and 16 kDa. The larger subunit appeared to be glycosylated and was identical to DoxA, and the smaller was identical to DoxD. Both subunits had been described previously as a part of the terminal quinol:oxygen oxidoreductase complex (cytochrome aa3).


Assuntos
Acidianus/enzimologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxirredutases/metabolismo , Oxigênio/metabolismo , Enxofre/metabolismo , Tiossulfatos/metabolismo , Acidianus/genética , Sequência de Aminoácidos , Membrana Celular/enzimologia , Ferricianetos/metabolismo , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona)/química , NAD(P)H Desidrogenase (Quinona)/genética , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Ácido Tetratiônico/metabolismo
18.
Biochim Biophys Acta ; 1655(1-3): 306-20, 2004 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-15100046

RESUMO

The membrane bound aa(3)-type quinol:oxygen oxidoreductase from the hyperthermophilic archaeon, Acidianus ambivalens, which thrives at a pH of 2.5 and a temperature of 80 degrees C, has several unique structural and functional features as compared to the other members of the heme-copper oxygen reductase superfamily, but shares the common redox-coupled, proton-pumping function. To better understand the properties of the heme a(3)-Cu(B) catalytic site, a resonance Raman spectroscopic study of the enzyme under a variety of conditions and in the presence of various ligands was carried out. Assignments of several heme vibrational modes as well as iron-ligand stretching modes are made to serve as a basis for comparing the structure of the enzyme to that of other oxygen reductases. The CO-bound oxidase has conformations that are similar to those of other oxygen reductases. However, the addition of CO to the resting enzyme does not generate a mixed valence species as in the bovine aa(3) enzyme. The cyanide complex of the oxidized enzyme of A. ambivalens does not display the high stability of its bovine counterpart, and a redox titration demonstrates that there is an extensive heme-heme interaction reflected in the midpoint potentials of the cyanide adduct. The A. ambivalens oxygen reductase is very stable under acidic conditions, but it undergoes an earlier alkaline transition than the bovine enzyme. The A. ambivalens enzyme exhibits a redox-linked reversible conformational transition in the heme a(3)-Cu(B) center. The pH dependence and H/D exchange demonstrate that the conformational transition is associated with proton movements involving a group or groups with a pK(a) of approximately 3.8. The observed reversibility and involvement of protons in the redox-coupled conformational transition support the proton translocation model presented earlier. The implications of such conformational changes are discussed in relation to general redox-coupled proton pumping mechanisms in the heme-copper oxygen reductases.


Assuntos
Acidianus/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/análogos & derivados , Monóxido de Carbono/metabolismo , Domínio Catalítico , Cianetos/metabolismo , Heme/química , Concentração de Íons de Hidrogênio , Ligantes , Oxirredução , Fotólise , Bombas de Próton/química , Bombas de Próton/metabolismo , Análise Espectral Raman
19.
Biochem J ; 381(Pt 1): 137-46, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15030315

RESUMO

The SOR (sulphur oxygenase reductase) is the initial enzyme in the sulphur-oxidation pathway of Acidianus ambivalens. Expression of the sor gene in Escherichia coli resulted in active, soluble SOR and in inclusion bodies from which active SOR could be refolded as long as ferric ions were present in the refolding solution. Wild-type, recombinant and refolded SOR possessed indistinguishable properties. Conformational stability studies showed that the apparent unfolding free energy in water is approx. 5 kcal x mol(-1) (1 kcal=4.184 kJ), at pH 7. The analysis of the quaternary structures showed a ball-shaped assembly with a central hollow core probably consisting of 24 subunits in a 432 symmetry. The subunits form homodimers as the building blocks of the holoenzyme. Iron was found in the wild-type enzyme at a stoichiometry of one iron atom/subunit. EPR spectroscopy of the colourless SOR resulted in a single isotropic signal at g=4.3, characteristic of high-spin ferric iron. The signal disappeared upon reduction with dithionite or incubation with sulphur at elevated temperature. Thus both EPR and chemical analysis indicate the presence of a mononuclear iron centre, which has a reduction potential of -268 mV at pH 6.5. Protein database inspection identified four SOR protein homologues, but no other significant similarities. The spectroscopic data and the sequence comparison led to the proposal that the Acidianus ambivalens SOR typifies a new type of non-haem iron enzyme containing a mononuclear iron centre co-ordinated by carboxylate and/or histidine ligands.


Assuntos
Proteínas de Bactérias/química , Heme , Ferro/metabolismo , Oxirredutases/química , Acidianus/enzimologia , Sequência de Aminoácidos/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas Arqueais/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Dimerização , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/genética , Microscopia Eletrônica/métodos , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases/ultraestrutura , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Conformação Proteica , Desnaturação Proteica/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/ultraestrutura , Especificidade por Substrato , Enxofre/metabolismo
20.
Appl Microbiol Biotechnol ; 64(5): 605-10, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-14997352

RESUMO

The 3-hydroxypropionate cycle, a pathway for autotrophic carbon dioxide fixation, is reviewed with special emphasis on the biochemistry of CO2 fixing enzymes in Acidianus brierleyi, a thermophilic and acidophilic archeon. In the 3-hydroxypropionate cycle, two enzymes, acetyl-CoA carboxylase and propionyl-CoA carboxylase, catalyze CO2 fixation. It has been shown in A. brierleyi, and subsequently in Metallosphaera sedula, that acetyl-CoA carboxylase is promiscuous, acting equally well on acetyl-CoA and propionyl-CoA. The subunit structure of the acyl-CoA carboxylase was shown to be alpha4beta4gamma4. Gene cloning revealed that the genes encoding the three subunits are adjacent to each other. accC encodes the beta-subunit (59 kDa subunit, biotin carboxylase subunit), accB encodes the gamma-subunit (20 kDa subunit, biotin carboxyl carrier protein), and pccB encodes the alpha-subunit (62 kDa subunit, carboxyltransferase subunit). Sequence analyses showed that accC and accB are co-transcribed and that pccB is transcribed separately. Potential biotechnological applications for the 3-hydroxypropionate cycle are also presented.


Assuntos
Acidianus/metabolismo , Dióxido de Carbono/metabolismo , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Acidianus/enzimologia , Acidianus/genética , Sequência de Aminoácidos , Sequência de Bases , Metilmalonil-CoA Descarboxilase/genética , Metilmalonil-CoA Descarboxilase/metabolismo , Dados de Sequência Molecular
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