RESUMEN
Hydrogenase-1 (HYD1), overexpressed by twofold, has been purified to homogeneity and to a high specific activity from a mutant strain (AP6) of Escherichia coli which lacks hydrogenase-2. Plasma emission spectroscopy indicated that 0.93 atom of nickel and 11.4 iron atoms were present in HYD1. EPR studies on the as isolated HYD1 detected a complex 3Fe-4S signal and a Ni(III) species. Reduction with hydrogen gas caused disappearance of both the 3Fe-4S cluster and initial Ni(III) signals. At the same time the EPR signature (small g = 2.19 signal) of the activated hydrogenase appeared. The detection of a 4Fe-4S cluster signal was noted. Reduction of HYD1 with sodium dithionite caused all nickel signals to disappear. The 4Fe-4S complex intensity was slightly increased. The EPR responses in the three oxidation-reduction states are consistent with other known (NiFe)-hydrogenases.
Asunto(s)
Escherichia coli/enzimología , Hidrogenasas/química , Ditionita/química , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/genética , Hidrogenasas/aislamiento & purificación , Mutación , Oxidación-ReducciónRESUMEN
We isolated seven different bacteria from rice seedlings grown from surface sterilized seeds. Three were associated with the rice seed husk and the other four were growing endophytically within the seed. Microscopic studies revealed that the endophytes were concentrated in the root stele region. Some of the bacteria exhibited strong anti-fungal activity against Rhizoctonia solani, Pythium myriotylum, Guamannomyces graminis and Heterobasidium annosum.
RESUMEN
The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced. They occur in an operon (hyb) which contains seven open reading frames. An hyb deletion mutant (strain AP3) failed to grown on dihydrogen-fumarate medium and also produced very low levels of HYD1. All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3. The hyb operon was mapped at 65 min on the E. coli chromosome.
Asunto(s)
Escherichia coli/genética , Hidrogenasas/genética , Operón , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Análisis Mutacional de ADN , Escherichia coli/enzimología , Genes Bacterianos , Prueba de Complementación Genética , Hidrogenasas/química , Hidrogenasas/metabolismo , Datos de Secuencia Molecular , Fenotipo , Análisis de Secuencia de ADNRESUMEN
Redox intermediates of D. desulfuricans ATCC 27774 [NiFe] hydrogenase were generated under dihydrogen. Detailed redox titrations, coupled to EPR measurements, give access to the mid-point redox potentials of the iron-sulfur centers and of the Nickel-B signal that represents the ready form of the enzyme. The interaction between the dihydrogen molecule and the nickel centre was probed by the observation of an isotopic effect on the EPR signals detected in turnover conditions, by comparison of the H2O/H2 and D2O/D2-reacted samples.
Asunto(s)
Desulfovibrio/enzimología , Hidrogenasas/química , Sitios de Unión , Desulfovibrio/genética , Campos Electromagnéticos , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , TemperaturaRESUMEN
Two electrophoretic forms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrio gigas have been detected by Western analysis. The faster moving form co-migrates with the large subunit from purified, active enzyme. Amino acid sequence and composition of the C-terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence. Processing of the nascent large subunit occurs by C-terminal cleavage between His536 and Val537, residues which are highly conserved among [NiFe] hydrogenases. Mutagenesis of the analogous residues, His582 and Val583, in the E. coli hydrogenase-1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.
Asunto(s)
Hidrogenasas/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Aminoácidos/análisis , Western Blotting , Cromatografía por Intercambio Iónico , Desulfovibrio/enzimología , Escherichia coli/enzimología , Hidrogenasas/química , Hidrogenasas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-DirigidaRESUMEN
The nature of the iron-sulfur clusters in oxidized and reduced forms of Fe-only hydrogenases from Desulfovibrio vulgaris, Thermotoga maritima, and Clostridium pasteurianum has been investigated by resonance Raman spectroscopy. The results indicate the presence of ferredoxin-like [4Fe-4S]2+,+ and [2Fe-2S]2+,+ clusters in both T. maritima hydrogenase and C. pasteurianum hydrogenase I, but only [4Fe-4S]2+,+ clusters in D. vulgaris hydrogenase. This necessitates a reevaluation of the iron-sulfur cluster composition of C. pasteurianum hydrogenase I and indicates that the resonance Raman bands in the oxidized hydrogenase that were previously attributed to the hydrogen activating center [Macor, K. A., Czernuszewicz, R. S., Adams, M. W. W., & Spiro, T. G. (1987) J. Biol. Chem. 262, 9945-9947] arise from an indigenous [2Fe-2S]2+ cluster. No resonance Raman bands that could be uniquely attributed to the oxidized or reduced hydrogen activating center were observed. This suggests that the hydrogen activating center is a novel Fe center that is unrelated to any known type of Fe-S cluster.
Asunto(s)
Clostridium/enzimología , Desulfovibrio vulgaris/enzimología , Bacterias Anaerobias Gramnegativas/enzimología , Hidrogenasas/química , Proteínas Hierro-Azufre/química , Hidrogenasas/clasificación , Hierro/química , Proteínas Hierro-Azufre/clasificación , Espectrometría Raman , Azufre/químicaRESUMEN
The enzymology of the heterodimeric (NiFe) and (NiFeSe) hydrogenases, the monomeric nickel-containing hydrogenases plus the multimeric F420-(NiFe) and NAD(+)-(NiFe) hydrogenases are summarized and discussed in terms of subunit localization of the redox-active nickel and non-heme iron clusters. It is proposed that nickel is ligated solely by amino acid residues of the large subunit and that the non-heme iron clusters are ligated by other cysteine-rich polypeptides encoded in the hydrogenase operons which are not necessarily homologous in either structure or function. Comparison of the hydrogenase operons or putative operons and their hydrogenase genes indicate that the arrangement, number and types of genes in these operons are not conserved among the various types of hydrogenases except for the gene encoding the large subunit. Thus, the presence of the gene for the large subunit is the sole feature common to all known nickel-containing hydrogenases and unites these hydrogenases into a large but diverse gene family. Although the different genes for the large subunits may possess only nominal general derived amino acid homology, all large subunit genes sequenced to date have the sequence R-X-C-X-X-C fully conserved in the amino terminal region of the polypeptide chain and the sequence of D-P-C-X-X-C fully conserved in the carboxyl terminal region. It is proposed that these conserved motifs of amino acids provide the ligands required for the binding of the redox-active nickel. The existing EXAFS (Extended X-ray Absorption Fine Structure) information is summarized and discussed in terms of the numbers and types of ligands to the nickel and the various redox species of nickel defined by EPR spectroscopy. New information concerning the ligands to nickel is presented based on site-directed mutagenesis of the gene encoding the large subunit of the (NiFe) hydrogenase-1 of Escherichia coli. Based on considerations of the biochemical, molecular and biophysical information, ligand environments of the nickel in different redox states of the (NiFe) hydrogenase are proposed.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/química , Hidrogenasas/química , Metaloproteínas/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Proteínas de Hierro no Heme , Relación Estructura-ActividadRESUMEN
Mössbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. Mössbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the Mössbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.
Asunto(s)
Desulfovibrio/enzimología , Hemo/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Nitrito Reductasas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Nitrito Reductasas/aislamiento & purificación , Oxidación-Reducción , Análisis EspectralRESUMEN
The intracellular location of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris was determined using pre-embedding and post-embedding immunoelectron microscopic procedures. Polyclonal antisera directed against the purified (NiFe) and (NiFeSe) hydrogenases were raised in rabbits. One-day-old cultures of D. vulgaris, grown on a lactate/sulfate medium, were used for all experiments in these studies. For post-embedding labeling studies cells were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde, dehydrated with methanol, and embedded in the low-temperature resin Lowicryl K4M. Our post-embedding studies using antibody-gold or protein-A-gold as electron-dense markers revealed the location of the two hydrogenases exclusively at the cell periphery; the precise membrane location was then demonstrated by pre-embedding labeling. Spheroplasts were incubated with the polyclonal antisera against (NiFe) and (NiFeSe) hydrogenase followed by ferritin-linked secondary antibodies prior to embedding and sectioning. The observed labeling pattern unequivocally revealed that the antigenic reactive sites of the (NiFe) hydrogenase are located in the near vicinity of the cytoplasmic membrane facing into the periplasmic space, whereas the (NiFeSe) hydrogenase is associated with the cytoplasmic side of the cytoplasmic membrane.
Asunto(s)
Desulfovibrio/enzimología , Hidrogenasas/análisis , Metales/análisis , Sitios de Unión , Membrana Celular/enzimología , Desulfovibrio/ultraestructura , Hidrogenasas/inmunología , Sueros Inmunes/inmunología , Inmunohistoquímica , Metales/inmunología , Microscopía Electrónica/métodos , Coloración y EtiquetadoRESUMEN
The soluble (cytoplasmic plus periplasmic) Ni/Fe-S/Se-containing hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from cells grown in an 57Fe-enriched medium, and its iron-sulfur centers were extensively characterized by Mössbauer and EPR spectroscopies. The data analysis excludes the presence of a [3Fe-4S] center, either in the native (as isolated) or in the hydrogen-reduced states. In the native state, the non-heme iron atoms are arranged as two diamagnetic [4Fe-4S]2+ centers. Upon reduction, these two centers exhibit distinct and unusual Mössbauer spectroscopic parameters. The centers were found to have similar mid-point potentials (approximately -315 mV) as determined by oxidation-reduction titratins followed by EPR.
Asunto(s)
Desulfovibrio/enzimología , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Metaloproteínas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Hidrogenasas/aislamiento & purificación , Proteínas Hierro-Azufre/aislamiento & purificación , Sustancias Macromoleculares , Níquel/análisis , Conformación Proteica , Selenio/análisis , Análisis Espectral/métodosRESUMEN
DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.
Asunto(s)
Clonación Molecular , Escherichia coli/genética , Hidrogenasas/genética , Operón , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Membrana Celular/enzimología , Escherichia coli/enzimología , Genes Bacterianos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Conformación Proteica , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). Mössbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical Mössbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.
Asunto(s)
Desulfovibrio/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Oxidorreductasas/aislamiento & purificación , Adenosina Monofosfato/farmacología , Sitios de Unión , Ditionita/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/análisis , Oxidación-Reducción , Oxidorreductasas/antagonistas & inhibidores , Espectrofotometría Ultravioleta , Espectroscopía de Mossbauer , Sulfitos/farmacología , TemperaturaRESUMEN
A hypothetical model of the complex formed between the iron-sulfur protein rubredoxin and the tetraheme cytochrome c3 from the sulfate-reducing bacteria Desulfovibrio vulgaris (Hildenborough) has been proposed utilizing computer graphic modeling, computational methods and NMR spectroscopy. The proposed complex appears feasible on the basis of complementary electrostatic interaction and steric factors and is consistent with the data from NMR experiments. In this model, the non-heme iron atom of rubredoxin is in close proximity to heme 1 of cytochrome c3. The complex is stabilized by charge-pair interactions and hydrogen bonds. This complex is compared to the flavodoxin-cytochrome c3 complex previously proposed [Stewart, D. E., LeGall, J., Moura, I., Moura, J. J. G., Peck, H. D. Jr, Xavier, A. V., Weiner, P. K. & Wampler, J. E. (1988) Biochemistry 27, 2444-2450] and new NMR data shows that both proteins interact with the same heme group of the cytochrome as postulated.
Asunto(s)
Grupo Citocromo c/metabolismo , Desulfovibrio/metabolismo , Ferredoxinas/metabolismo , Rubredoxinas/metabolismo , Sulfatos/metabolismo , Sitios de Unión , Gráficos por Computador , Desulfovibrio/enzimología , Transporte de Electrón , Transferencia de Energía , Flavodoxina/análisis , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oxidación-ReducciónRESUMEN
The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and Mössbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.
Asunto(s)
Desulfovibrio/enzimología , Hidrogenasas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Hierro/análisis , Proteínas Hierro-Azufre/metabolismo , Cinética , Níquel/análisis , Oxidación-Reducción , Análisis EspectralRESUMEN
The nucleotide sequences encoding the [NiFe] hydrogenase from Desulfovibrio gigas and the [NiFeSe] hydrogenase from Desulfovibrio baculatus (N.K. Menon, H.D. Peck, Jr., J. LeGall, and A.E. Przybyla, J. Bacteriol. 169:5401-5407, 1987; C. Li, H.D. Peck, Jr., J. LeGall, and A.E. Przybyla, DNA 6:539-551, 1987) were analyzed by the codon usage method of Staden and McLachlan. The reported reading frames were found to contain regions of low codon probability which are matched by more probable sequences in other frames. Renewed nucleotide sequencing showed the probable frames to be correct. The corrected sequences of the two small and large subunits share a significant degree of sequence homology. The small subunit, which contains 10 conserved cysteine residues, is likely to coordinate at least 2 iron-sulfur clusters, while the finding of a selenocysteine codon (TGA) near the 3' end of the [NiFeSe] large-subunit gene matched by a regular cysteine codon (TGC) in the [NiFe] large-subunit gene indicates the presence of some of the ligands to the active-site nickel in the large subunit.
Asunto(s)
Desulfovibrio/genética , Genes Bacterianos , Hidrogenasas/genética , Secuencia de Aminoácidos , Codón , ADN Bacteriano/genética , Hierro , Metaloproteínas/genética , Datos de Secuencia Molecular , Níquel , Selenio , Homología de Secuencia de Ácido NucleicoRESUMEN
The effect of acetylene on the activity of the three types of hydrogenase from the anaerobic sulfate reducing bacteria has been investigated. The (Fe) hydrogenase is resistant to inhibition by acetylene while the nickel-containing hydrogenases are inhibited by acetylene with the (NiFe) hydrogenase being 10-50 fold more sensitive than the (NiFeSe) hydrogenase. In addition the Ni(III) EPR signal (g approximately 2.3) of the "as isolated" (NiFe) hydrogenase was significantly decreased in intensity upon exposure to acetylene.
Asunto(s)
Acetileno/farmacología , Desulfovibrio/enzimología , Euryarchaeota/enzimología , Hidrogenasas/metabolismo , Desulfovibrio/efectos de los fármacos , Espectroscopía de Resonancia por Spin del Electrón , Euryarchaeota/efectos de los fármacos , Hidrogenasas/antagonistas & inhibidoresRESUMEN
Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Euryarchaeota/metabolismo , Metano/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Isótopos de Carbono , Espectroscopía de Resonancia Magnética/métodos , FósforoRESUMEN
The periplasmic hydrogenase containing equivalent amounts of nickel and selenium plus non-heme iron [NiFeSe) hydrogenase) has been purified from cells of the sulfate reducing bacterium Desulfovibrio baculatus (DSM 1748) grown on a lactate/sulfate medium containing natural Se isotopes and the nuclear isotope, 77Se. Both the 77Se-enriched and unenriched hydrogenases were shown to be free of other hydrogenases and characterized with regard to their Se contents. EPR studies of the reduced nickel signal generated by redox titrations of the enriched and unenriched (NiFeSe) hydrogenases demonstrated that the gx = 2.23 and gy = 2.17 resonances are appreciably broadened by the spin of the 77Se nucleus (I = 1/2). This observation demonstrates unambiguously that the unpaired electron is shared by the Ni and Se atoms and that Se serves as a ligand to the nickel redox center of the (NiFeSe) hydrogenase.
Asunto(s)
Desulfovibrio/enzimología , Hidrogenasas/aislamiento & purificación , Níquel/análisis , Selenio/análisis , Sitios de Unión , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia por Spin del Electrón , Hidrogenasas/metabolismo , Cinética , LigandosRESUMEN
Ni and Se x-ray absorption spectroscopic studies of the [NiFeSe]hydrogenases from Desulfovibrio baculatus are described. The Ni site geometry is pseudo-octahedral with a coordinating ligand composition of 3-4 (N,O) at 2.06 A, 1-2 (S,Cl) at 2.17 A, and 1 Se at 2.44 A. The Se coordination environment consists of 1 C at 2.0 A and a heavy scatterer M (M = Ni or Fe) at approximately 2.4 A. These results are interpreted in terms of a selenocysteine residue coordinated to the Ni site. The possible role of the Ni-Se site in the catalytic activation of H2 is discussed.
Asunto(s)
Cisteína/análogos & derivados , Desulfovibrio/enzimología , Hidrogenasas/metabolismo , Metaloendopeptidasas/metabolismo , Níquel , Selenio , Sitios de Unión , Hierro/metabolismo , Oxidación-Reducción , Conformación Proteica , Selenio/metabolismo , Selenocisteína , Análisis EspectralRESUMEN
Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)