Onthe ironsulfur cluster in hydrogenase fromclostridium. Overexpression of a hydrogenase gene in clostridium. The fermentation of glycerol by clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. The most recent reports of quantitative fe and s analysis indicate that this hydrogenase contains 20. Pdf fehydrogenase is a distinct class of hydrogenproducing metalloenzyme, present in a wide. Coupled ferredoxin and crotonyl coenzyme a coa reduction. A threedimensional structure for the monomeric ironcontaining hydrogenase cpi from clostridium pasteurianum was. Hydrogenase 1 was also shown to reduce a 2nitroimidazole misonidazole and a 4nitroimidazole in the presence of its required electron carriers. Xray crystal structure of the feonly hydrogenase cpi from. A number of clostridial species are also known to degrade and ferment various biomass. Fermentation of glycerol by clostridium pasteurianum batch.
The ironsulfur centers and the function of hydrogenase. When cultures were grown in medium with glucose, 1. Homologous overexpression of hydrogenase and glycerol. Here, we demonstrate that substitution of any of these residues resulted in a drastic. Mechanism of proton transfer in fefehydrogenase from.
Xray crystal structure of the feonly hydrogenase cpi. Advanced paramagnetic resonance studies of the feonly hydrogenase i from clostridium pasteurianum cpi joshua telser 1,2, paul m. Some aspects of hydrogenase activity and nitrogen fixation in. Metal analyses showed that neither hydrogenase contains nickel or any other met als in significant amounts. Highlypurified bidirectional hydrogenase hydrogenase 1 of clostridiwn pasteurianum could rapidly reduce several 2, 4 and 5nitroimidazole compounds via an electron carriercoupled mechanism. The physical and catalytic properties of hydrogenase ii of clostridium pasteurianum. A threedimensional structure for the monomeric ironcontaining hydrogenase cpi from clostridium pasteurianum was determined to 1. 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.
Peck and gest 1 reported that crude extracts of clostridium butylicum produce small amounts of hydrogen from aqueous dithionite hydrosulfite. Fermentation of mixed substrates by clostridium pasteurianum. Clostridium pasteurianum is a bacterium that can metabolize glycerol anaerobically as sole carbon and energy source, producing a unique product pro. The physiological functions and structural determinants of. 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. Ironlimitation caused lactate production 38 mol100 mol from glucose in batch and continuous culture.
The major examples of fefe hydrogenase studied to date are from the green microalgae chlamydomonas reinhardtii crhyda1. 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. 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. An investigation of hydrogenase i and hydrogenase ii from clostridium pasteurianum by resonance raman spectroscopy. The pyruvic dehydrogenase system of clostridium pasteurianum has been shown to catalyze a multistep reaction. Parametersaffecting solvent production by clostridium. The physiological functions and structural determinants of catalytic bias in the fefehydrogenases cpi and cpii of clostridium pasteurianum. Hydrogenases from azotobacter vinelandii and clostridium pasteurianum reduced methylene blue, ferricyanide, benzyl and methylviologens when hydrogen was the donor. Structural similarities between the nterminal domain of clostridium pasteurianum hydrogenase and planttype ferredoxins. It was the first free living nonsymbiotic microorganism discovered that could fix free nitrogen from the air. Pdf primary structure of hydrogenase from clostridium. Clostridium pasteurianum previously known as clostridium pastorianum is a bacterium discovered in 1890 by the russian microbiologist sergei winogradsky. This has led to intense research focusing on use of fefe hydrogenase for sustainable production of h 2. Transcript mapping of the rubredoxin gene from clostridium pasteurianum.
Clostridium pasteurianum is a bacterium that can metabolize glycerol anaerobically as sole carbon. Molecular dynamics and experimental investigation of h2. Iron hydrogenase 1 clostridium pasteurianum uniprot. A, crystal structure of the fefehydrogenase from c.
A putative butyrate kinase gene buk is adjacent to the hyda gene. The first generation of biochemical studies of complex, ironsulfurclustercontaining fefehydrogenases and monitrogenase were carried out on enzymes purified from clostridium pasteurianum strain w5. Fefehydrogenase structure and putative proton transfer pathway. 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. Ironsulfur clusters of hydrogenase i and hydrogenase ii. Pdf an investigation of hydrogenase i and hydrogenase ii. Previously we have shown that dual substrate fermentation using glucose and glycerol enhanced the cell growth and butanol production significantly.
Ironlimitation caused lactate production 38 mol100 mol from glucose in. 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. 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. A, crystal structure of the fefe hydrogenase from c. Hydrogenase i1 of clostridium pasteurianum is a monomeric protein of mr 53,000 containing 8 iron and 8 acidlabile sulfide atomsmol. 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. 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.
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. The journal of biological chemistry 0 1984 by the american society of biological chemists, inc. International hydrogenases conference 2004 81 table 1 comparison of algal and bacterial fehydrogenase o2 sensitivities fehydrogenase ic50 value s c. Pdf role of fehydrogenase in biological hydrogen production. Three of the four presumed catalytic intermediates h ox, h red h red and h sred were characterized, using various spectroscopic techniques. 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. 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. The ironsulfur centers and the function of hydrogenase from. Cloning and sequencing of the gene encoding the 2fe2s ferredoxin from clostridium pasteurianum.
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. Spectrophotometric experiments with aged and dialyzed preparations have implicated flavin, the hydrogenase system, and iron in this reaction. It has the ability to convert carbohydrates to butyrate, acetate. The hydrogenase system of clostridium pasteurianum journal of. The physical and catalytic properties of hydrogenase i1 of clostridium pasteurianum a comparison with hydrogenase i received for publication, january 10, 1984. In 189395 he also discovered clostridium pasteurianum, an anaerobic bacterium i.
Advanced paramagnetic resonance studies of the feonly. Some aspects of hydrogenase activity and nitrogen fixation. Hydrogenase i1 of clostridium pasteurianum is a monomeric protein of mr 53,000 containing 8. Examples include the hydrogenases from algae, such as chlamydomonas reinhardtii, and clostridial species, such as clostridium pasteurianum. Occurrence nickel in carbonmonoxide dehydrogenase clostridium. U09982 clostridium pasteurianum atcc 60 spo0a spo0a gene, complete cds. A more detailed discussion on the physiological role of hydrogenase among microorganisms appeared recently mortenson and chen, 1974.
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. However, the majority of the nickel in cell extracts was found to electrophorese independently of co dehydrogenase. The hydrogenase system of clostridium pasteurianum r. Under phosphate limitation, glucose was fermentedalmostexclusively to acetate andbutyrateindependentlyofthe phandgrowthrate. It is distinct from hydrogenase i from the same organism mr 60,000 12 fe and 12 szmol. The active site of the diiron hydrogenase is known as the hcluster. On the ironsulfur cluster in hydrogenase from clostridium. Isolation and properties of a unidirectional h2oxidizing hydrogenase from the strictly anaerobic n2fixing bacterium clostridium pasteurianum w5.
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. 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. Fefehydrogenases cpi and cpii of clostridium pasteurianum. Hughes press preparations of either organism in tris 2amino2hydroxymethyl propane1. The xray crystal structure of rubredoxin from clostridium pasteurianum. A putative hydrogenase hyda gene of clostridium perfringens encodes a protein with strong identity to clostridium pasteurianum hydrogenase i. Clostridium pasteurianum is a strictly anaerobic, grampositive. 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. 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. The metal cofactor containing active site of the fefehydrogenases. H 2 turnover at the fefehydrogenase cofactor hcluster is assumed to follow.
Fefe hydrogenase structure and putative proton transfer pathway. Clostridium pasteurianum is becoming increasingly attractive for the production of chemicals and fuels such as nbutanol and 1,3propanediol. Other articles where clostridium pasteurianum is discussed. Fermentation of glycerol by clostridium pasteurianum. 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. More than 60 gl glycerol was utilized, and up to 17 gl. The major examples of fefehydrogenase studied to date are from the green microalgae chlamydomonas reinhardtii crhyda1. Fefe and nifehydrogenase diversity, mechanism, and maturation. The physical and catalytic properties of hydrogenase i1 of. Overexpression of dhad1 and dhak enhanced glycerol uptake and h 2 yield by 1. Freeenergy calculation studies show that the side chains of two conserved glutamate residues, glu279 and glu282. 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. Previous studies suggested that two distinct fefehydrogenases are expressed differentially under nitrogenfixing and nonnitrogenfixing conditions.
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. Complete activity profile of clostridium acetobutylicum fefe. Primary structure of hydrogenase from clostridium pasteurianum. Clostridium pasteurianum 574 fd, fv, plant fd 6064. 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. Esearch rticles xray crystal structure of the structure. Jun 21, 2016 clostridium pasteurianum is becoming increasingly attractive for the production of chemicals and fuels such as nbutanol and 1,3propanediol. Ironsulfur clusters of hydrogenase i and hydrogenase ii of.
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