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1.
Upon exposure to CO during anaerobic growth, the purple phototrophic bacterium Rhodospirillum rubrum expresses a CO-oxidizing H(2) evolving enzymatic system. The CO-oxidizing enzyme, carbon monoxide dehydrogenase (CODH), has been purified and extensively characterized. However the electron transfer pathway from CODH to the CO-induced hydrogenase that evolves H(2) is not well understood. CooF is an Fe-S protein that is the proposed mediator of electron transfer between CODH and the CO-induced hydrogenase. Here we present the spectroscopic and biochemical properties of the CODH:CooF complex. The characteristic EPR signals observed for CODH are largely insensitive to CooF complexation. Metal analysis and EPR spectroscopy show that CooF contains 2 Fe(4)S(4) clusters. The observation of 2 Fe(4)S(4) clusters for CooF contradicts the prediction of 4 Fe(4)S(4) clusters based on analysis of the amino acid sequence of CooF and structural studies of CooF homologs. Comparison of in vivo and in vitro CO-dependent H(2) evolution indicates that approximately 90% of the activity is lost upon cell lysis. We propose that the loss of two labile Fe-S clusters from CooF during cell lysis may be responsible for the low in vitro CO-dependent H(2) evolution activity. During the course of these studies, a new assay for CODH:CooF was developed using membranes from an R. rubrum mutant that did not express CODH:CooF, but expressed high levels of the CO-induced hydrogenase. The assay revealed that the CO-induced hydrogenase requires the presence of CODH:CooF for optimal H(2) evolution activity. 相似文献
2.
Eight Ni proteins are known and three of these, CO dehydrogenase (CODH), acetyl-CoA synthase (ACS), and hydrogenase, are Ni-Fe-S proteins. In the last three years, the long-awaited structures of CODH and ACS have been solved. The bioinorganic community was shocked, as the structures of the active sites of CODH and ACS, the C- and A-cluster, respectively, which each had been predicted to consist of a [Fe4S4] cluster bridged to a single Ni, revealed unexpected compositions and arrangements. Crystal structures of ACS revealed major differences in protein conformation and in A-cluster composition; for example, a [Fe4S4] cluster bridged to a binuclear center in which one of the metal binding sites was occupied by Ni, Cu, or Zn. Recent studies have revealed Ni-Ni to be the active state, unveiled the source of the heterogeneity that had plagued studies of CODH/ACS for decades, and produced a metal-replacement strategy to generate highly active and nearly homogeneous enzyme.Abbreviations CFeSP corrinoid iron-sulfur protein - CH3H4folate methyltetrahydrofolate - CODH/ACS carbon monoxide dehydrogenase/acetyl-CoA synthases - ENDOR electron nuclear double resonance - MeTr methyltransferase 相似文献
3.
Won Bae Jeon Steven W. Singer Paul W. Ludden Luis M. Rubio 《Journal of biological inorganic chemistry》2005,10(8):903-912
Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO2. A unique [NiFe4S4] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation
of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH•Ni complex prior to conversion
to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination
of the [Fe3S4] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are
important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new
variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the
apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in
which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step. 相似文献
4.
Takahiro Inoue Kyousuke Takao Yuto Fukuyama Takashi Yoshida 《Bioscience, biotechnology, and biochemistry》2013,77(4):582-587
Carbon monoxide dehydrogenase-I (CODH-I) from the CO-utilizing bacterium Carboxydothermus hydrogenoformans are expected to be utilized as a part of reproducible carbon dioxide photoreduction system. However, the over-expression system for CODH-I remains to be constructed. CODH-I constitutes a hydrogenase/CODH gene cluster including a gene encoding a Ni-insertion accessory protein, CooC (cooC3). Through co-expression of CooC3, we found an over-expression system with higher activity. The Rec-CODH-I with the co-expression exhibits 8060?U/mg which was approximately threefold than that without co-expression (2270?U/mg). In addition, co-expression resulted in Ni2+ content increase; the amount of Ni atoms of Rec-CODH-I was approximately thrice than that without co-expression. 相似文献
5.
Twenty species and strains of aerobic CO-oxidizing bacteria were screened for the occurrence of plasmids. Six of them harbored plasmids between 45 and 558kb. Megaplasmids of 428 and 558 kb were resolved in Alcaligenes carboxydus. Restriction digest patterns of plasmids from different carboxydotrophic bacteria were dissimilar. However, the patterns obtained with the plasmids from the strains OM5, OM4 and OM2 of Pseudomonas carboxydovorans were very much the same. The nine cured mutants of P. carboxydovorans OM5, as well as the deletion mutant OM5-29, could not grow chemolithotrophically with CO or H2 plus CO2, as they were devoid of CO dehydrogenase, hydrogenase and ribulose bisphosphate carboxylase. The deletion mutant OM5-24 retained the ability to grow with CO. It could not grow with H2 plus CO2 and was devoid of H2ase. The data suggest the residence of structural and/or regulatory genes of CODH, H2ase and RuBPCx on plasmid pHCG3 of P. carboxydovorans.Abbreviations CODH
carbon monoxide dehydrogenase
- CRM
cross reacting material
- EMS
ethyl methane sulfonate
- H2ase
hydrogenase
- kb
kilobase
- NTG
N-methyl-N-nitro-N-nitrosoguanidine
- RuBPCx
ribulose bisphosphate carboxylase
- SDS
sodium dodecylsulfate 相似文献
6.
7.
E C Hatchikian M Bruschi J Le Gall 《Biochemical and biophysical research communications》1978,82(2):451-461
The hydrogenase of the sulfate-reducer has been purified to homogeneity. The pure enzyme shows a specific activity of 90 μmoles H2 evolved/min./mg protein. Its molecular weight is 89,500 and its is composed of two different subunits (mol. wt. : 62,000 and 26,000) which are not covalently bound. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein. The millimolar extinction coefficients of the hydrogenase are 46.5 and 170 respectively at 400 and 280 nm. It contains about 12 iron atoms and 12 acid-labile sulfur groups per molecule and the quantitative extrusion of the Fe-S centers of the hydrogenase indicates the presence of 3 Fe4S4 clusters. This hydrogenase has 21 half-cystine residues per molecule and a preponderance of aromatic amino-acids. 相似文献
8.
Jiann-Shin Chen D.K. Blanchard 《Biochemical and biophysical research communications》1984,122(1):9-16
Clostridium pasteurianum has two distinct hydrogenases, the bidirectional hydrogenase and the H2-oxidizing (uptake) hydrogenase. The H2-oxidizing hydrogenase has been purified (up to 970-fold) to a specific activity of 17,600 μmol H2 oxidized/min·mg protein (5 mM methylene blue) or 3.5 μmol H2 produced/min·mg protein (1 mM methyl viologen). The uptake hydrogenase has a Mr of 53,000 (one polypeptide chain). Depending upon how protein was measured, the Fe and S= contents (gatom/mol) were 4.7 and 5.2 (by the dye-binding assay) or 7.2 and 8.0 (by the Lowry method). Both reduced and oxidized forms of the enzyme gave electron paramagnetic resonance signals. The activation energy for H2-production and H2-oxidation by the uptake hydrogenase was 59.1 and 31.2 kJ/mol, respectively. In the exponential phase of growth, the ratio of uptake hydrogenase/bidirectional hydrogenase in NH3-grown cells was much lower than that in N2-fixing cells. 相似文献
9.
Charles Gauquelin Carole Baffert Pierre Richaud Emma Kamionka Emilien Etienne David Guieysse Laurence Girbal Vincent Fourmond Isabelle André Bruno Guigliarelli Christophe Léger Philippe Soucaille Isabelle Meynial-Salles 《BBA》2018,1859(2):69-77
The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_ΔDA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H+/H2 potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin. 相似文献
10.
Abstract Clostridium thermoaceticum was cultivated heterotrophically under CO2 , carbon monoxide (CO), and H2 gas phases. Formate dehydrogenase (FDH) levels increased 4-fold in CO cultures; formyltetrahydrofolate synthetase (FTS) levels were not influenced by the cultivation gas phases tested. While CO dehydrogenase (CODH) was slightly stimulated in CO cultures, CO-dependent acetyl phosphate-synthesizing system (APSS) activity increased sharply in both CO and H2 cultures. Y glucose values increased, whereas doubling times and acetate to biomass ratios decreased significantly in CO cultures, suggesting that CO cultures were energetically dissimilar to non-CO cultures. This finding, together with the absence of CO-dependent ATP-independent synthesis of formyltetrahydrofolate (formyl-THF), supports the hypothesis that conservation of CO-derived energy involves electron transport phosphorylation. 相似文献
11.
Hideaki Ogata 《Journal of molecular biology》2010,402(2):428-10843
The crystal structure of the membrane-associated [NiFe] hydrogenase from Allochromatium vinosum has been determined to 2.1 Å resolution. Electron paramagnetic resonance (EPR) and Fourier transform infrared spectroscopy on dissolved crystals showed that it is present in the Ni-A state (> 90%). The structure of the A. vinosum [NiFe] hydrogenase shows significant similarities with [NiFe] hydrogenase structures derived from Desulfovibrio species. The amino acid sequence identity is ∼ 50%. The bimetallic [NiFe] active site is located in the large subunit of the heterodimer and possesses three diatomic non-protein ligands coordinated to the Fe (two CN− , one CO). Ni is bound to the protein backbone via four cysteine thiolates; two of them also bridge the two metals. One of the bridging cysteines (Cys64) exhibits a modified thiolate in part of the sample. A mono-oxo bridging ligand was assigned between the metal ions of the catalytic center. This is in contrast to a proposal for Desulfovibrio sp. hydrogenases that show a di-oxo species in this position for the Ni-A state. The additional metal site located in the large subunit appears to be a Mg2+ ion. Three iron-sulfur clusters were found in the small subunit that forms the electron transfer chain connecting the catalytic site with the molecular surface. The calculated anomalous Fourier map indicates a distorted proximal iron-sulfur cluster in part of the crystals. This altered proximal cluster is supposed to be paramagnetic and is exchange coupled to the Ni3+ ion and the medial [Fe3S4]+ cluster that are both EPR active (S = 1/2 species). This finding of a modified proximal cluster in the [NiFe] hydrogenase might explain the observation of split EPR signals that are occasionally detected in the oxidized state of membrane-bound [NiFe] hydrogenases as from A. vinosum. 相似文献
12.
Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system. 总被引:1,自引:1,他引:0
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R L Kerby S S Hong S A Ensign L J Coppoc P W Ludden G P Roberts 《Journal of bacteriology》1992,174(16):5284-5294
A 3.7-kb DNA region encoding part of the Rhodospirillum rubrum CO oxidation (coo) system was identified by using oligonucleotide probes. Sequence analysis of the cloned region indicated four complete or partial open reading frames (ORFs) with acceptable codon usage. The complete ORFs, the 573-bp cooF and the 1,920-bp cooS, encode an Fe/S protein and the Ni-containing carbon monoxide dehydrogenase (CODH), respectively. The four 4-cysteine motifs encoded by cooF are typical of a class of proteins associated with other oxidoreductases, including formate dehydrogenase, nitrate reductase, dimethyl sulfoxide reductase, and hydrogenase activities. The R. rubrum CODH is 67% similar to the beta subunit of the Clostridium thermoaceticum CODH and 47% similar to the alpha subunit of the Methanothrix soehngenii CODH; an alignment of these three peptides shows relatively limited overall conservation. Kanamycin cassette insertions into cooF and cooS resulted in R. rubrum strains devoid of CO-dependent H2 production with little (cooF::kan) or no (cooS::kan) methyl viologen-linked CODH activity in vitro, but did not dramatically alter their photoheterotrophic growth on malate in the presence of CO. Upstream of cooF is a 567-bp partial ORF, designated cooH, that we ascribe to the CO-induced hydrogenase, based on sequence similarity with other hydrogenases and the elimination of CO-dependent H2 production upon introduction of a cassette into this region. From mutant characterizations, we posit that cooH and cooFS are not cotranscribed. The second partial ORF starts 67 bp downstream of cooS and would be capable of encoding 35 amino acids with an ATP-binding site motif. 相似文献
13.
The influence of reduced sulfur compounds (including stored S0) on H2 evolution/consumption reactions in the purple sulfur bacterium, Thiocapsa roseopersicina BBS, was studied using mutants containing only one of the three known [NiFe] hydrogenase enzymes: Hox, Hup or Hyn. The observed
effects depended on the kind of hydrogenase involved. The mutant harbouring Hox hydrogenase was able to use S2O32−, SO32−, S2− and S0 as electron donors for light-dependent H2 production. Dark H2 evolution from organic substrates via Hox hydrogenase was inhibited by S0. Under light conditions, endogenous H2 uptake by Hox or Hup hydrogenases was suppressed by S compounds. СО2-dependent H2 uptake by Hox hydrogenase in the light required the additional presence of S compounds, unlike the Hup-mediated process.
Dark H2 consumption via Hyn hydrogenase was connected to utilization of S0 as an electron acceptor and resulted in the accumulation of H2S. In wild type BBS, with high levels of stored S0, dark H2 production from organic substrates was significantly lower, but H2S accumulation significantly higher, than in the mutant GB1121(Hox+). There is a possibility that H2 produced via Hox hydrogenase is consumed by Hyn hydrogenase to reduce S0. 相似文献
14.
Properties of the solubilized membrane-bound hydrogenase from the photosynthetic bacterium Rhodospirillum rubrum. 总被引:7,自引:0,他引:7
The membrane-bound hydrogenase of the photosynthetic bacterium Rhodospirillum rubrum has been purified 490-fold with a yield of 5.8%. The enzyme was homogeneous by disc gel electrophoresis. A method for the permanent, oxygen-insensitive, staining of hydrogenase on polyacrylamide gels is described. The enzyme is a monomer of molecular weight about 66,000 containing four iron and four acid-labile sulfur atoms per molecule. The electron paramagnetic resonance spectrum at 20 °K exhibits a strong signal in the oxidized state only with g > 2—this is characteristic of high potential iron-sulfur protein. The hydrogenase is thermostable and also resistant to both denaturation agents and oxygen inactivation. Carbon monoxide reversibly inhibits the enzyme but metal-complexing and thiol-blocking reagents have little effect on activity. The enzyme will catalyze both H2 evolution and H2 uptake in the presence of many artificial electron carriers but the two activities differ in their pH optima. There is a correlation between H2 evolution activity and the redox potential of the mediator dye. Ferredoxins and pyridine nucleotides do not readily interact with the hydrogenase. We have shown that irradiation of a solution containing methyl viologen, EDTA, proflavin, and R. rubrum hydrogenase will evolve hydrogen continuously for over 9 h. However, the enzyme evolves hydrogen at only very low rates from in vitro chloroplast-ferredoxin and chloroplast-methyl viologen systems. R. rubrum hydrogenase has a number of properties in common with the hydrogenases purified from two other photosynthetic bacteria, Chromatium and Thiocapsa, but is distinct from the hydrogenases of nonphotosynthetic bacteria. 相似文献
15.
Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme. 总被引:2,自引:2,他引:0
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In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing. 相似文献
16.
Michael Cunliffe 《The ISME journal》2011,5(4):685-691
The Marine Roseobacter Clade (MRC) is a numerically and biogeochemically significant component of the bacterioplankton. Annotation of multiple MRC genomes has revealed that an abundance of carbon monoxide dehydrogenase (CODH) cox genes are present, subsequently implying a role for the MRC in marine CO cycling. The cox genes fall into two distinct forms based on sequence analysis of the coxL gene; forms I and II. The two forms are unevenly distributed across the MRC genomes. Most (18/29) of the MRC genomes contain only the putative form II coxL gene. Only 10 of the 29 MRC genomes analysed have both the putative form II and the definitive form I coxL. None have only the form I coxL. Genes previously shown to be required for post-translational maturation of the form I CODH enzyme are absent from the MRC genomes containing only form II. Subsequent analyses of a subset of nine MRC strains revealed that only MRC strains with both coxL forms are able to oxidise CO. 相似文献
17.
Two ferredoxins from nitrogen-fixing cells of the phototrophic bacterium Rhodopseudomonas capsulata, strain B10, are purified to a homogeneous state and characterized. The molecular mass of ferredoxin I is about 12 kDa and that of ferredoxin II, 18 kDa. Ferredoxin I contains 8 Fe2+ and 8 S2?; ferredoxin II has 4 Fe2+ and 4 S2? per molecule. The redox potential of ferredoxin I is about ?270 mV and that of ferredoxin II ?419 mV. Ferredoxin I is more labile to the action of O2, O?2, H2O2 and heating. The ferredoxins are also different in their absorption and EPR spectra, amino acid composition and electron-transfer activity to Rps. capsulata nitrogenase: both C2H2 reduction and H2 evolution by Rps. capsulata nitrogenase proceed faster in the presence of ferredoxin I than in case of ferredoxin II. Synthesis of ferredoxin I takes place only in Rps. capsulata nitrogen-fixing cells grown in light under anaerobic conditions whereas ferredoxin II formation does not depend on the source of nitrogen or the growth medium, though the amount of ferredoxin II varies with the growth conditions. Its highest level has been found in the cells grown in lactate-limited medium in the presence of CO2 and light or in the presence of glutamate in darkness under anaerobic conditions. 相似文献
18.
《BBA》2020,1861(7):148188
Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO2 as a source of carbon. The recent detailed characterizations of some of them have evidenced a great diversity in terms of catalytic properties and resistance to O2. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp. AM4 (Tc). We have also crystallized CooS2, which is coupled in vivo to a hydrogenase. CooS1 and CooS2 are homodimers, and harbour five metalloclusters: two [Ni4Fe-4S] C clusters, two [4Fe-4S] B clusters and one interfacial [4Fe-4S] D cluster. We show that both are dependent on a maturase, CooC1 or CooC2, which is interchangeable. The homologous protein CooC3 does not allow Ni insertion in either CooS. The two CODHs from Tc have similar properties: they can both oxidize and produce CO. The Michaelis constants (Km) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO2. Product inhibition is observed only for CO2 reduction, consistent with CO2 binding being much weaker than CO binding. The two enzymes are rather O2 sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O2 than any other CODH for which these data are available. 相似文献
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