Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.12.7.2 (hydrogenase)
3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The uptake hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) from the bacteroids of soybean root nodules infected with Rhizobium japonicum 110 has been purified and characterized. Bacteroids were prepared, then broken by sonication. The particulate enzyme was solubilized by treatment with Triton X-100 and further purified by polyethylene glycol fractionation, DEAE-cellulose and Sephadex G-100 chromatography. The specific activity has been increased 196-fold to 19.6 units/mg protein. The molecular weight is 63 300 as determined by gel filtration and 65 300 as determined by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The enzyme is O2 sensitive, with a half-life of 70 min when exposed to air. The pH optimum of the solubilized enzyme is near 5.5; the Km for H2 is 1.4 microM. Suitable electron acceptors are methylene blue, ferricyanide, 2,6-dichlorophenolindophenol, and cytochrome c. Benzyl viologen is reduced slowly; methyl viologen, NAD(P)+, FAD, FMN, and O2 are not reduced. The optimum temperature for activity is 65-70 degrees C with an activation energy of 9.2 kcal. H2 evolution by the enzyme has been demonstrated. The hydrogenase is well-suited to function in an environment where all the available H2 is generated in situ.
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PMID:Purification and properties of the particulate hydrogenase from the bacteroids of soybean root nodules. 4 Jun 1

Hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) catalyzes three types of reactions, i.e., (1) conversion between hydrogen modifications, para-H2 and ortho-H2, (2) exchange reaction between hydrogen isotopes, and (3) reversible oxidoreduction of an electron carrier with H2 and protons. We observed that purified desulfovibrio hydrogenase in the dry state could catalyze not only the conversion and exchange reactions (Yagi, T., Tsuda, M., Mori, Y. and Inokuchi, H. (1969) J. Am. Chem. Soc. 91, 2801) but also the reversible oxidoreduction of the electron carrier, cytochrome c3 with H2. The rate of the conversion was in the range from 0.1 to 0.65 mol H2 converted per mol hydrogenase per s, and the ratio of the conversion rate to the exchange rate was near 5. The rate of oxidoreduction of cytochrome c3 in the dry state was 0.015 mol H2 taken up in the forward reaction and 0.003 mol H2 released in the reverse reaction per mol hydrogenase per s. The process of these reactions could be explained by the observations that the hydrogenase molecule in the dry state has protons which are directly exchangeable with H2 during catalytic process. The reversible oxidoreduction of cytochrome c3 is also explained by inter- and intramolecular electron transfer among cytochrome c3 molecules.
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PMID:Hydrogenase activity in the dry state: isotope exchange and reversible oxidoreduction of cytochrome c3. 22 28

Cytochrome c-553 of Desulfovibrio vulgaris, Miyazaki, was purified to homogeneity. The absorption spectrum of the ferro form has four peaks at 553, 525, 417 and 317 nm with a plateau near 280 nm, and that of the ferri form has three peaks at 525, 410 and 360 nm with a plateau near 280 nm and a shoulder at 560 nm. The millimolar absorbance coefficient of the alpha-peak of the ferro form is 23.9. The molecular weight of cytochrome c-553 is 8000, and it contains one heme. Its isoelectric point is rather alkaline, and its standard redox potential is -0.26 V at pH 7.0. Its amino acid composition is unique; it lacks proline, isoleucine and tryptophan. Ferrocytochrome c-553 does not combine with CO, nor does it transfer electrons directly to various redox carriers such as flavin nucleotides, methylene blue, indigodisulfonate, 5-methylphenazinium methyl sulfate, 1-methoxy-5-methylphenazinium methyl sulfate, viologens and cytochrome c3, but is oxidized by ferricyanide or by O2. Cytochrome c-553 can be reduced by formate dehydrogenase of this bacterium in the presence of formate, but not by hydrogenase under H2. The formate dehydrogenase does not reduce cytochrome c3 in the presence of formate. The systematic name for formate dehydrogenase of D. vulgaris is, therefore, established as formate:ferricytochrome c-553 oxidoreductase in EC subclass 1.22.-.
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PMID:Purification and properties of cytochrome c-553, an electron acceptor for formate dehydrogenase of Desulfovibrio vulgaris, Miyazaki. 22 35

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.
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PMID:Factor 420-dependent pyridine nucleotide-linked hydrogenase system of Methanobacterium ruminantium. 23 34

Methanobacterium ruminantium was shown to possess a formate dehydrogenase which is linked to factor 420 (F420) as the first low-molecular-weight or anionic electron transfer coenzyme. Reduced F420 obtained from the formate dehydrogenase can be further linked to the formation of hydrogen via the previously described F420-dependent hydrogenase reaction, thus constituting an apparently simple formate hydrogenlyase system, or to the reduction of nicotinamide adenine dinucleotide phosphate via F420:nicotinamide adenine dinucleotide phosphate oxidoreductase. The results indicate that hydrogen and formate, the only known energy sources for M. ruminantium and many other methanogenic bacteria, should be essentially equivalent as sources of electrons in the metabolism of this organism.
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PMID:Factor 420-dependent pyridine nucleotide-linked formate metabolism of Methanobacterium ruminantium. 23 35

A survey on organisms able to use molecular hydrogen as electron donor in the energy-yielding process is presented. In the group of the aerobic hydrogen-oxidizing bacteria so far two types of hydrogenases have been encountered, a NAD-reducing, soluble enzyme (H2 : NAD oxidoreductase) and a membrane-bound enzyme unable to reduce pyridine nucleotides. With respect to the distribution of both types of hydrogenases three groups of hydrogen-oxidizing bacteria can be diffentiated containing (i) both types (Alcaligenes eutrophus), (ii) a soluble enzyme only (Nocardia opaca lb), and (iii) a membrane-bound hydrogenase only (majority of genera and species). The results of studies on the NAD-specific hydrogenase of A. eutrophus are summarized. Results on the solubilization and purification of the membrane-bound hydrogenase of A. eutrophus are presented in detail. The enzyme was solubilized from purified membranes by Triton X-100 and sodium desoxycholate or phospholipase D. The crude membrane extract was fractionated by ammonium sulfate precipitation and chromatography on carboxymethylcellulose at pH 5.5. The enzyme was stable in potassium phosphate buffer; it resembles the soluble enzyme with respect to stability under oxidizing conditions. Further biochemical and immunological data indicate, however, that both enzymes are different with respect to their native structure.
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PMID:Hydrogen metabolism in aerobic hydrogen-oxidizing bacteria. 66 83

The sulfur-reducing bacterium Spirillum 5175 was investigated with regard to membrane constituents that might be part of the sulfur oxidoreductase which converts elemental sulfur to hydrogen sulfide. Regardless of the electron acceptor used for cultivation of the bacteria, i.e. elemental sulfur, fumarate, or nitrate (Sp. 5175S,F,N), the qualitative pattern of cytochromes and Fe-S proteins did not change significantly, as documented by ultraviolet/visible and electron paramagnetic resonance spectroscopy of oxidized (as isolated) and reduced (dithionite) samples. With elemental sulfur the prominent cytochrome exhibited absorption maxima at 553, 522.5 and 426 nm in the reduced state. In fumarate-grown cells two prominent cytochromes were found with maxima at 561, 551, 530, 521 and 430 nm. Two b-type cytochromes with Em at -198 mV and -20 mV vs the standard hydrogen electrode were identified in the membrane fraction of Sp. 5175F. A yellow pigment was extracted and identified as a flexirubin-type pigment. Although present in large quantities, it seemed not to be involved in the reduction of elemental sulfur. Menaquinone, MK 6 (Mr 580) was the prominent quinone identified in Sp. 5175. Characterization of a second quinone was not attempted because of its much lower concentration. The membrane constituents of Sp. 5175 were solubilized by a variety of detergents and detergent mixtures. A colorimetric procedure with photochemically reduced phenosafranin as the electron donor and cysteamine trisulfide (RS-S-SR, R = -CH2CH2NH2) as the electron acceptor was used to detect sulfur oxidoreductase activity. Three membrane proteins of Sp. 5175 were purified: (1) an [NiFe] hydrogenase, homogeneous by SDS/polyacrylamide gel electrophoresis, with electron paramagnetic resonance signals as isolated at gx,y,z = 2.01, 2.16, 2.33 (100 K), and a strong signal at g = 2.02 below 20 K; (2) a cytochrome b, Fe-S-dependent fumarate reductase, and (3) a protein apparently linked to the sulfur oxidoreductase activity. In contrast to fumarate reductase, no b-type cytochrome was present in the fractions exhibiting sulfur oxidoreductase activity. The presence of Fe-S centers was demonstrated by electron paramagnetic resonance spectroscopy at 10 K. It is not clear whether the c-type cytochrome in the same fractions is part of the sulfur-reducing apparatus of Sp. 5175.
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PMID:Investigations on microbial sulfur respiration. Isolation, purification, and characterization of cellular components from Spirillum 5175. 184 72

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum.
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PMID:Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes. 199 10

The proposal that nitrite exerts its inhibitory effect on anaerobic bacteria by direct interaction with the iron-sulphur proteins of the phosphoroclastic system was investigated. The effects of nitrate, nitrite with or without ascorbate, and nitric oxide on the growth of Clostridium sporogenes in liquid cultures at pH 7.4, on the rates of hydrogen production, and on the activities of the enzymes pyruvate-ferredoxin oxidoreductase and hydrogenase, and of ferredoxin were investigated. In agreement with previous studies, nitrate was the least effective inhibitor of cell growth, and nitric oxide the most effective. Nitrite reductase activity was very low in C. sporogenes, indicating that the presence of external reducing agents would be necessary for the reduction of nitrite to nitric oxide. Inhibition by nitrite was enhanced by ascorbate; 0.5 mM-nitrite with 10 mM-ascorbate stopped growth completely. In partially-purified preparations 4.1 mM-NaNO2 and equimolar ascorbate caused complete inactivation of hydrogenase activity but only partial (up to 78%) inactivation of pyruvate-ferredoxin oxidoreductase. This agreed with the loss of hydrogen production observed with nitrite in vivo. Inhibition occurred within 5 min, and was irreversible in each case. Electron paramagnetic resonance (EPR) spectroscopy showed that paramagnetic [Fe(NO)2(SR)2] species were formed during growth in the presence of nitrite, and were associated with cells. However, the intensity of these EPR signals did not correlate with the inhibition of cell growth. The [4Fe-4S] clusters in ferredoxin were shown by EPR spectroscopy to be resistant to treatment with 3.6 mM-NaNO2 and 3.6 mM-ascorbate. It is concluded that the effects of nitrite on pre-formed iron-sulphur proteins are not convincing as a basis for the lethal effects on bacterial cells.
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PMID:Electron paramagnetic resonance spectroscopic investigation of the inhibition of the phosphoroclastic system of Clostridium sporogenes by nitrite. 217 68

Certain reagents, such as ascorbate or iron salts and thiols, enhance the bacteriostatic action of nitrite on food-spoilage bacteria. This may be due to the formation of nitric oxide and iron-thiol-nitrosyl [( Fe-S-NO]) complexes. The minimum concentrations of these reagents required to inhibit growth of Clostridium sporogenes were investigated. A mixture of nitrite (0.72 mM) with iron (1.44 mM) and cysteine (2.16 mM) was found to be extremely inhibitory when autoclaved and diluted into the culture medium. This mixture caused rapid cessation of growth and loss of cell viability at a final concentration corresponding to 40 microM-nitrite. If added to the initial culture medium, it prevented growth at 5 microM-nitrite. The mixture was more inhibitory, on the basis of the nitrite concentration used, than the 'Perigo factor', obtained by autoclaving nitrite in growth medium. [Fe-S-NO] compounds of known chemical structure were tested to determine if they were responsible for this effect. Total inhibition of cell growth was observed with the tetranuclear clusters [Fe4S3(NO)7] (Roussin's black salt), [Fe4S4(NO)4] or [Fe4Se3(NO)7], added at concentrations equivalent to 10 microM-nitrite, or with [Fe2(SMe)2(NO)4] (methyl ester of Roussin's red salt), equivalent to 200 microM-nitrite. The rate of hydrogen production in growing cell cultures was inhibited by [Fe4S3(NO)7] at levels equivalent to 2.5 microM-nitrite. EPR spectra of the inhibited cells showed features with g-values of 2.03, characteristic of mononuclear iron-nitrosyl species, and, under non-reducing conditions, an unusual signal at g = 1.65. There was no correlation between growth inhibition and the g = 2.03 signal, though there was a better correlation between inhibition and the g = 1.65 signal. The direct effects of the compounds were tested on the iron-sulphur proteins of the phosphoroclastic system, namely ferredoxin, pyruvate-ferredoxin oxidoreductase and hydrogenase. EPR spectra and enzyme assays showed that these proteins were not destroyed by [Fe4S3(NO)7], [Fe4S4(NO)4], [Fe2(SMe)2(NO)4], [Fe(SPh)2(NO)2], or M2 (an autoclaved mixture of 66 mM-cysteine, 3.6 mM-FeSO4 and 0.72 mM-NaNO2) at concentrations higher than those that caused total inhibition of cell growth. Inhibition of cells by [Fe-S-NO] compounds is unlikely to be due to interaction with the preformed enzymes. The possible formation of iron-nitrosyl complexes in vivo, and their inhibitory actions, are discussed.
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PMID:Interactions of iron-thiol-nitrosyl compounds with the phosphoroclastic system of Clostridium sporogenes. 217 69


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