When cultures were grown in medium with glucose, 1. Hydrogenase, purified to an average specific activity of 328 mumol of h2 evolvedmin x mg of protein from clostridium pasteurianum w5, was found to have 45 fe and 45 labile sulfur atoms per molecule of 60,000 molecular weight, in. Fefe hydrogenase structure and putative proton transfer pathway. Primary structure of hydrogenase from clostridium pasteurianum. Pdf role of fehydrogenase in biological hydrogen production.
Ironsulfur clusters of hydrogenase i and hydrogenase ii. The iron and acidlabile sulfide contents and the electron paramagnetic resonance epr properties of hydrogenase i bidirectional and hydrogenase ii uptake of clostridium pasteurianum strain w5 have been determined on the basis of quantitative amino acid analyses. Abstract hydrogenase, purified to an average specific activity of 328 imol of h2 evolvedmin x mg of protein from clostridium pasteurianum w5, was found to have 45 fe and 45 labile sulfur atoms per molecule of 60,000 molecular weight, in contrast with earlier reports of 12 fe per molecule. A hydrogenase is an enzyme that catalyses the reversible oxidation of molecular hydrogen. More than 60 gl glycerol was utilized, and up to 17 gl. Cloning and sequencing of the gene encoding the 2fe2s ferredoxin from clostridium pasteurianum. Occurrence nickel in carbonmonoxide dehydrogenase clostridium. Transcript mapping of the rubredoxin gene from clostridium pasteurianum. Fefe and nifehydrogenase diversity, mechanism, and maturation. Fermentation of glycerol by clostridium pasteurianum batch. Molecular dynamics and experimental investigation of h2. The most recent reports of quantitative fe and s analysis indicate that this hydrogenase contains 20.
Advanced paramagnetic resonance studies of the feonly hydrogenase i from clostridium pasteurianum cpi joshua telser 1,2, paul m. Advanced paramagnetic resonance studies of the feonly. Hydrogenase, purified to an average specific activity of 328 mumol of h2 evolvedmin x mg of protein from clostridium pasteurianum w5, was found to have 45 fe and 45 labile sulfur atoms per molecule of 60,000 molecular weight, in contrast with earlier reports of 12 fe per molecule. Clostridium pasteurianum is a strictly anaerobic, grampositive. The hydrogenase system of clostridium pasteurianum journal of. Structural similarities between the nterminal domain of clostridium pasteurianum hydrogenase and planttype ferredoxins. Spectrophotometric experiments with aged and dialyzed preparations have implicated flavin, the hydrogenase system, and iron in this reaction. Freeenergy calculation studies show that the side chains of two conserved glutamate residues, glu279 and glu282. H 2 turnover at the fefe hydrogenase cofactor hcluster is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydridospecies which again is doubleoxidized to release another proton. Fermentation of glycerol by clostridium pasteurianum. A more detailed discussion on the physiological role of hydrogenase among microorganisms appeared recently mortenson and chen, 1974. U09982 clostridium pasteurianum atcc 60 spo0a spo0a gene, complete cds.
Hydrogenase i1 of clostridium pasteurianum is a monomeric protein of mr 53,000 containing 8 iron and 8 acidlabile sulfide atomsmol. Information about hydrogenase genes and their products has been reported from a few clostridia, for example, fehydrogenase i of clostridium pasteurianum, hydrogenase a of clostridium perfringens, and hydrogenase a of clostridium acetobutylicum p262. Ironlimitation caused lactate production 38 mol100 mol from glucose in. On the ironsulfur cluster in hydrogenase from clostridium. Examples include the hydrogenases from algae, such as chlamydomonas reinhardtii, and clostridial species, such as clostridium pasteurianum. Fefehydrogenases cpi and cpii of clostridium pasteurianum. This has led to intense research focusing on use of fefe hydrogenase for sustainable production of h 2. The latter is particularly important for obligate anaerobes such as clostridium pasteurianum. Although codehydrogenase has not been purified from clostridium pasteurianum, this clostridium wasearlier shownto oxidize coto co27, andevidence wasrecently presented by diekert et al. Clostridium pasteurianum 574 fd, fv, plant fd 6064.
Fermentation of mixed substrates by clostridium pasteurianum. Clostridium pasteurianum is a bacterium that can metabolize glycerol anaerobically as sole carbon. A putative butyrate kinase gene buk is adjacent to the hyda gene. It was the first free living nonsymbiotic microorganism discovered that could fix free nitrogen from the air clostridium pasteurianum is a producer of carboxylic acids. A, crystal structure of the fefe hydrogenase from c. International hydrogenases conference 2004 81 table 1 comparison of algal and bacterial fehydrogenase o2 sensitivities fehydrogenase ic50 value s c. Clostridium pasteurianum previously known as clostridium pastorianum is a bacterium discovered in 1890 by the russian microbiologist sergei winogradsky. Xray crystal structure of the feonly hydrogenase cpi from. The physical and catalytic properties of hydrogenase i1 of. Pdf primary structure of hydrogenase from clostridium. The physical and catalytic properties of hydrogenase i1 of clostridium pasteurianum a comparison with hydrogenase i received for publication, january 10, 1984.
Abstract hydrogenase, purified to an average specific activity of 328 jsmol of h2evolvedmin xmgofprotein from clostridium pasteurianum w5,was found to have 45 feand45labile sulfur atomspermoleculeof60,000molecularweight, in contrast withearlier reports of 12fepermolecule. Peck and gest 1 reported that crude extracts of clostridium butylicum produce small amounts of hydrogen from aqueous dithionite hydrosulfite. Xray crystal structure of the feonly hydrogenase cpi. The physiological functions and structural determinants of catalytic bias in the fefehydrogenases cpi and cpii of clostridium pasteurianum. The protein is a monomer separated into a catalytic domain containing the 6fe6s hcluster and three nterminal domains that in total contain three 4fe4s clusters and a 2fe2s cluster. The fermentation of glycerol by clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. The xray crystal structure of rubredoxin from clostridium pasteurianum.
Three of the four presumed catalytic intermediates h ox, h red h red and h sred were characterized, using various spectroscopic techniques. The ironsulfur centers and the function of hydrogenase. To better understand the proton transport through the h2 production catalysts, the fefe hydrogenases, we have undertaken a modeling and simulation study of the proton transfer processes mediated by amino acid sidechain residues in hydrogenase i from clostridium pasteurianum. Autotrophicus and fefe hydrogenase from clostridium pasteurianum by arti sharma pandey a dissertation submitted in partial fulfillment of the requirements of the degree of doctor of philosophy in biochemistry montana state university bozeman, montana july 2007. A putative hydrogenase hyda gene of clostridium perfringens encodes a protein with strong identity to clostridium pasteurianum hydrogenase i. Some aspects of hydrogenase activity and nitrogen fixation in. The major examples of fefehydrogenase studied to date are from the green microalgae chlamydomonas reinhardtii crhyda1. Isolation and properties of a unidirectional h2oxidizing hydrogenase from the strictly anaerobic n2fixing bacterium clostridium pasteurianum w5. The metal cofactor containing active site of the fefehydrogenases. Esearch rticles xray crystal structure of the structure. It is distinct from hydrogenase i from the same organism mr 60,000 12 fe and 12 szmol. In 189395 he also discovered clostridium pasteurianum, an anaerobic bacterium i. Hydrogenase 1 was also shown to reduce a 2nitroimidazole misonidazole and a 4nitroimidazole in the presence of its required electron carriers. It was the first free living nonsymbiotic microorganism discovered that could fix free nitrogen from the air.
The first generation of biochemical studies of complex, ironsulfurclustercontaining fefehydrogenases and monitrogenase were carried out on enzymes purified from clostridium pasteurianum strain w5. Onthe ironsulfur cluster in hydrogenase fromclostridium. A number of clostridial species are also known to degrade and ferment various biomass. Pdf fehydrogenase is a distinct class of hydrogenproducing metalloenzyme, present in a wide. Under phosphate limitation, glucose was fermentedalmostexclusively to acetate andbutyrateindependentlyofthe phandgrowthrate. In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3propanediol, butyric and acetic acids and ethanol. A computational analysis of this pathway in the fefehydrogenase from clostridium pasteurianum revealed that the solventexposed residue of the pathway glu282 forms hydrogen bonds to two. Cpi, an enzyme that catalyzes the twoelectron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of. Homologous overexpression of hydrogenase and glycerol. Clostridium pasteurianum an overview sciencedirect topics. However, the majority of the nickel in cell extracts was found to electrophorese independently of co dehydrogenase. Here, we demonstrate that substitution of any of these residues resulted in a drastic. Hydrogenase 1 was also shown to reduce a 2nitroimidazole misonidazole and a 4nitroimidazole in the presence of its required electron carriers including ferredoxin, the flavin coenzymes. Overexpression of dhad1 and dhak enhanced glycerol uptake and h 2 yield by 1.
Hydrogenase i1 of clostridium pasteurianum is a monomeric protein of mr 53,000 containing 8. Hughes press preparations of either organism in tris 2amino2hydroxymethyl propane1. Clostridium pasteurianum is becoming increasingly attractive for the production of chemicals and fuels such as nbutanol and 1,3propanediol. Metal analyses showed that neither hydrogenase contains nickel or any other met als in significant amounts. The physical and catalytic properties of hydrogenase ii of clostridium pasteurianum. Other articles where clostridium pasteurianum is discussed. Overexpression of a hydrogenase gene in clostridium. Iron hydrogenase 1 clostridium pasteurianum uniprot.
Clostridium pasteurianum is a bacterium that can metabolize glycerol anaerobically as sole carbon and energy source, producing a unique product pro. A, crystal structure of the fefehydrogenase from c. Complete activity profile of clostridium acetobutylicum fefe. Cpi, an enzyme that catalyzes the twoelectron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of protein. Coupled ferredoxin and crotonyl coenzyme a coa reduction. A threedimensional structure for the monomeric ironcontaining hydrogenase cpi from clostridium pasteurianum was. The pyruvic dehydrogenase system of clostridium pasteurianum has been shown to catalyze a multistep reaction. It has the ability to convert carbohydrates to butyrate, acetate.
Fefehydrogenase structure and putative proton transfer pathway. Hydrogenases from azotobacter vinelandii and clostridium pasteurianum reduced methylene blue, ferricyanide, benzyl and methylviologens when hydrogen was the donor. H 2 turnover at the fefehydrogenase cofactor hcluster is assumed to follow. H 2 turnover at the fefehydrogenase cofactor hcluster is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydridospecies which again is doubleoxidized to release another proton. Nov 04, 2011 previously, structural characterization of fefe hydrogenase from clostridium pasteurianum indicated a potential proton transport pathway involving four residues cys299, glu279, ser319, and glu282 that connect the active site to the enzyme surface. Previously we have shown that dual substrate fermentation using glucose and glycerol enhanced the cell growth and butanol production significantly. The journal of biological chemistry 0 1984 by the american society of biological chemists, inc. The hydrogenase system of clostridium pasteurianum r. A threedimensional structure for the monomeric ironcontaining hydrogenase cpi from clostridium pasteurianum was determined to 1. Jun 21, 2016 clostridium pasteurianum is becoming increasingly attractive for the production of chemicals and fuels such as nbutanol and 1,3propanediol. The major examples of fefe hydrogenase studied to date are from the green microalgae chlamydomonas reinhardtii crhyda1. Jul 12, 2017 the first generation of biochemical studies of complex, ironsulfurclustercontaining fefehydrogenases and monitrogenase were carried out on enzymes purified from clostridium pasteurianum strain w5.
The ironsulfur centers and the function of hydrogenase from. An investigation of hydrogenase i and hydrogenase ii from clostridium pasteurianum by resonance raman spectroscopy. Abstract hydrogenase, purified to an average specific activity of 328 mumol of h2 evolvedmin x mg of protein from clostridium pasteurianum w5, was found to have 45 fe and 45 labile sulfur atoms per molecule of 60,000 molecular weight, in contrast with earlier reports of 12 fe per molecule. The active site of the diiron hydrogenase is known as the hcluster. Parametersaffecting solvent production by clostridium. Information about hydrogenase genes and their products has been reported from a few clostridia, for example, fe hydrogenase i of clostridium pasteurianum, hydrogenase a of clostridium perfringens, and hydrogenase a of clostridium acetobutylicum p262. Hoffman 2 1chemistry program, roosevelt university, chicago, il 60605 usa, 2 department of chemistry, northwestern university, evanston, il 60208 usa, and 3 department of chemistry and. Mechanism of proton transfer in fefehydrogenase from. Highlypurified bidirectional hydrogenase hydrogenase 1 of clostridiwn pasteurianum could rapidly reduce several 2, 4 and 5nitroimidazole compounds via an electron carriercoupled mechanism. A computational analysis of this pathway in the fefe hydrogenase from clostridium pasteurianum revealed that the solventexposed residue of the pathway glu282 forms hydrogen bonds to two. Ironlimitation caused lactate production 38 mol100 mol from glucose in batch and continuous culture. The carbon monoxide co dehydrogenase activity band from clostridium pasteurianum was shown to contain nickel by in situ activity staining of polyacrylamide gels. Ironsulfur clusters of hydrogenase i and hydrogenase ii of.
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