Properties of the particulate enzyme F420-reducing hydrogenase isolated from Methanospirillum hungatei

1987 ◽  
Vol 33 (10) ◽  
pp. 896-904 ◽  
Author(s):  
G. Dennis Sprott ◽  
Kathleen M. Shaw ◽  
Terry J. Beveridge
Keyword(s):  
Gel Filtration ◽  
Methanobacterium Thermoautotrophicum ◽  
Methanosarcina Barkeri ◽  
Nonheme Iron ◽  
Anaerobic Conditions ◽  
Hydrophobic Properties ◽  
Iron Protein ◽  
Methanospirillum Hungatei ◽  
Cytoplasmic Membranes ◽  
Spectrum Characteristic

F420-reducing hydrogenase was isolated from spheroplast lysates of Methanospirillum hungatei by sedimentation, followed by sucrose gradient centrifugations and gel filtration. These procedures resulted in a preparation free of methyl reductase and cytoplasmic membranes. The hydrogenase was a brown protein with an absorption spectrum characteristic for a nonheme iron protein. In electron micrographs it was a coin-shaped, multisubunit protein complex of 15.9 nm diameter with a central depression on one surface. On phenyl Sepharose chromatography the hydrogenase exhibited hydrophobic properties. The holoenzyme was about 720 kilodaltons (kDa), composed of 50.7 and 30.7 kDa subunits in a ratio of 1:3. Each enzyme particle contained 6 or 7 Ni2+ atoms. H2-dependent reduction of F420 activity was readily, but transiently, reactivated by anaerobic conditions following exposure of the enzyme to air. Mg2+ or Ca2+ were stimulatory, but added FAD was not required. Antibody raised against the purified hydrogenase of strain GP1 gave a negative reaction with extracts of nine other methanogens and a reaction of identity with strain JF1 and Methanosarcina barkeri MS. Direct comparisons with the hyrogenase from Methanobacterium thermoautotrophicum ΔH revealed striking differences in subunit composition and in the acidity of the holoenzyme.

Investigation of serine hydroxymethyltransferase in methanogens

1998 ◽  
Vol 44 (7) ◽  
pp. 652-656 ◽  
Author(s):  
Zhaosheng Lin ◽  
Richard Sparling
Keyword(s):  
Key Words ◽  
Pyridoxal Phosphate ◽  
Methanobacterium Thermoautotrophicum ◽  
Methanosarcina Barkeri ◽  
Serine Hydroxymethyltransferase ◽  
Methanococcus Thermolithotrophicus ◽  
Cofactor Specificity ◽  
Methanospirillum Hungatei ◽  
Methanosphaera Stadtmanae ◽  
Methanosaeta Concilii

The cofactor specificity of serine hydroxymethyltransferase (SHMT) activities was tested in extracts of several methanogens using tetrahydromethanopterin (H4MPt) from Methanobacterium thermoautotrophicum Marburg, tetrahydrosarcinapterin (H4SPt) from Methanosarcina barkeri, and tetrahydrofolate (H4folate) as the potential C1 carrier. In Methanosphaera stadtmanae and Methanococcus thermolithotrophicus, the activities were H4MPt dependent. In Methanospirillum hungatei GP1, Methanosaeta concilii, Methanolobus tindarius, and Methanosarcina barkeri Fusaro, the activities were strictly H4folate dependent. H4SPt was reactive with the SHMT of Methanosphaera stadtmanae but not with that of Methanosarcina barkeri. In both Methanosarcina barkeri and Methanospirillum hungatei, pyridoxal phosphate stimulated SHMT activity. The apparent Km values for H4folate and L-serine were 0.086 and 0.29 mM in Methanosarcina barkeri and 0.065 and 0.31 mM in Methanospirillum hungatei, respectively. Key words: tetrahydromethanopterin, tetrahydrofolate, Archaea, serine hydroxymethyltransferase, methanogen.

Inhibition of methanogenesis in pure cultures by ammonia, fatty acids, and heavy metals, and protection against heavy metal toxicity by sewage sludge

1987 ◽  
Vol 33 (6) ◽  
pp. 551-554 ◽  
Author(s):  
Ken F. Jarrell ◽  
Michelle Saulnier ◽  
Art Ley
Keyword(s):  
Heavy Metals ◽  
Fatty Acids ◽  
Heavy Metal ◽  
Sewage Sludge ◽  
Metal Toxicity ◽  
Heavy Metal Toxicity ◽  
Methanosarcina Barkeri ◽  
Methanospirillum Hungatei ◽  
Nickel Zinc ◽  
Pure Cultures

The effect of ammonium chloride, sodium butyrate, sodium propionate, and the heavy metals nickel, zinc, and copper on methanogenesis by pure cultures of Methanospirillum hungatei, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobacterium formicicum at pH 6.5 was studied. The latter three strains were resistant to > 60 g/L of the volatile fatty acids and to > 10 g/L of NH3 N. Methanospirillum hungatei was somewhat more sensitive with 50% inhibition of methanogenesis occurring at 4.2 g/L NH3 N, 27 g/L butyrate, and 41 g/L propionate. All strains were very sensitive to both copper (1–5 mg/L) and zinc (1–10 mg/L), but much more resistant to nickel. Zinc and copper concentrations 30 to 270 times higher were required to cause inhibition of Msp. hungatei incubated in sewage sludge compared with buffer, indicating a strong protective environment was afforded the methanogens against heavy metal toxicity in the sludge.

Site of action of nonheme iron in the malate (flavine adenine dinucleotide) pathway of Mycobacterium phlei

1976 ◽  
Vol 22 (7) ◽  
pp. 1054-1057 ◽  
Author(s):  
A. K. Tyagi ◽  
T. L. Prasada Reddy ◽  
T. A. Venkitasubramanian
Keyword(s):  
Electron Transport ◽  
Ultraviolet Light ◽  
Nonheme Iron ◽  
Paramagnetic Resonance ◽  
Iron Protein ◽  
Mycobacterium Phlei ◽  
Diphenyl Tetrazolium Bromide ◽  
Electron Paramagnetic ◽  
Soluble Components ◽  
Malate Oxidation

Irradiation with ultraviolet light (360 nm) of cell-free extracts, electron-transport particles, and soluble components from Mycobacterium phlei resulted in the loss of malate oxidation by the flavine adenine dinucleotide pathway both in cell-free extracts and reconstituted systems. Addition of vitamin K1 restored the loss to the extent of 14% and 11% in cell-free extracts and reconstituted systems respectively. Electron-transport particles from M. phlei upon reduction with malate exhibited electron-paramagnetic resonance signals at g = 2.002 and 1.94, characteristic of napthosemiquinone and nonheme iron protein, respectively. Upon irradiating the particles with ultraviolet light (360 nm) these signals were not observed. Particulate flavine-adenine-dinucleotide-dependent malate dehydrogenase (EC 1.1.1.37) of M. phlei assayed by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide and phenazine methosulfate–2,6-dichlorophenolindophenol systems, which trap electrons at cytochrome c and at the flavine level respectively, was inhibited by o-phenanthroline. These observations suggest that nonheme iron protein is sensitive to ultraviolet light (360 nm) and participates before or in combination with flavine in the malate (flavine adenine dinucleotide) pathway of M. phlei.

Redesigning the Blue Copper Azurin into a Redox-Active Mononuclear Nonheme Iron Protein: Preparation and Study of Fe(II)-M121E Azurin

2014 ◽  
Vol 136 (35) ◽  
pp. 12337-12344 ◽  
Author(s):  
Jing Liu ◽  
Katlyn K. Meier ◽  
Shiliang Tian ◽  
Jun-long Zhang ◽  
Hongchao Guo ◽  
...  
Keyword(s):  
Nonheme Iron ◽  
Protein Preparation ◽  
Blue Copper ◽  
Iron Protein ◽  
Redox Active

Mammalian dihydroorotate – ubiquinone reductase complex. II. Correlation with cytochrome oxidase, mode of linkage with the cytochrome chain, and general properties

1969 ◽  
Vol 47 (7) ◽  
pp. 725-734 ◽  
Author(s):  
Richard W. Miller ◽  
Jack R. Curry
Keyword(s):  
Cytochrome Oxidase ◽  
Cytochrome B ◽  
Sucrose Gradient ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Mitochondrial Enzymes ◽  
Anaerobic Conditions ◽  
Beef Liver ◽  
And Behavior

Evidence is presented showing that a dihydroorotate-oxidizing system sediments with mitochondrial enzymes of beef liver in a sucrose gradient. Spectrophotometric experiments now indicate that dihydroorotate is as effective as succinate in reducing the cytochrome b content of whole and sonically disrupted mitochondria. The pyrimidine precursor also reduces the entire cytochrome chain under anaerobic conditions. Additional characterization of the dihydroorotate – ubiquinone reductase complex was carried out with regard to pH and temperature stability, and optima, metal inhibition, and behavior in gel filtration chromatography.

Aerobic purification of N5,N10-methylenetetrahydromethanopterin dehydrogenase, separated from N5,N10-methenyltetrahydromethanopterin cyclohydrolase, from Methanobacterium thermoautotrophicum strain Marburg

1989 ◽  
Vol 35 (4) ◽  
pp. 499-507 ◽  
Author(s):  
Biswarup Mukhopadhyay ◽  
Lacy Daniels
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Dehydrogenase Activity ◽  
Specific Activity ◽  
Methanobacterium Thermoautotrophicum ◽  
Anaerobic Conditions ◽  
Cell Extracts ◽  
First Time ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

The N5,N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain Marburg has been purified with reasonable yield and much higher specific activity than previously reported. For the first time it has been shown that both N5,N10-methylenetetrahydromethanopterin dehydrogenase and N5,N10-methenyltetrahydromethanopterin cyclohydrolase activities were stable under air and could be purified using aerobic operations. The dehydrogenase activity from Methanobacterium thermoautotrophicum Marburg was stable in phosphate buffer with or without glycerol or ammonium sulfate under both aerobic and anaerobic conditions. However, the presence of either 2-mercaptoethanol or dithiothreitol in the enzyme solution destroyed the enzyme activity during both aerobic and anaerobic incubations. Dehydrogenase was purified 62-fold using Phenyl-Sepharose and DEAE-Sephadex chromatography in succession under air. Both of these chromatographic methods separated dehydrogenase activity from N5,N10-methenyltetrahydromethanopterin cyclohydrolase; DEAE-Sephadex provided the best separation. Phenyl-Sepharose chromatography of the supernatant of cell extracts containing ammonium sulfate at 60% of saturation provided a 4.7-fold purification and 98% recovery of cyclohydrolase; this result established the air stability of N5,N10-methenyltetrahydromethanopterin cyclohydrolase from Methanobacterium thermoautotrophicum Marburg.Key words: methylenetetrahydromethanopterin dehydrogenase, methenyltetrahydromethanopterin cyclohydrolase, Methanobacterium, aerobic purification, oxygen stability.

scholarly journals Isolation, in the intact state, of the pterin molybdenum cofactor from xanthine oxidase

1989 ◽  
Vol 263 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J Deistung ◽  
R C Bray
Keyword(s):  
Aqueous Solution ◽  
Ion Exchange ◽  
Xanthine Oxidase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Molybdenum Cofactor ◽  
Anaerobic Conditions ◽  
Intact State

A procedure is described for isolation of the pterin molybdenum cofactor, in the active molybdenum-containing state, starting from purified milk xanthine oxidase. The method depends on the use of anaerobic-glove-cabinet techniques and on working in aqueous solution, in the presence of 1 mM-Na2S2O4. SDS was used to denature the protein, followed by ion-exchange chromatography and gel filtration. The cofactor, obtained at concentrations up to 0.5-1.0 mM, was fully active in the nit-1 assay [Hawkes & Bray (1984) Biochem. J. 214, 481-493], with a specific activity of 22 nmol of NO2-/min per pg-atom of Mo (with 15% molybdate-dependence). The Mr, determined by gel filtration, was about 610, consistent with the structure proposed by Kramer, Johnson, Ribeiro, Millington & Rajagopalan [(1987) J. Biol. Chem. 262, 16357-16363]. At pH 5.9, under anaerobic conditions, the cofactor was stable for at least 300 h at 20-25 degrees C.

Methanogenesis in the absence of intracytoplasmic membranes

1984 ◽  
Vol 30 (5) ◽  
pp. 594-604 ◽  
Author(s):  
G. D. Sprott ◽  
L. C. Sowden ◽  
J. R. Colvin ◽  
K. F. Jarrell ◽  
T. J. Beveridge
Keyword(s):  
Membrane System ◽  
Accurate Method ◽  
Methanobacterium Thermoautotrophicum ◽  
Methane Formation ◽  
Bacterial Cells ◽  
Optimal Temperature ◽  
Intracytoplasmic Membrane ◽  
Methanospirillum Hungatei ◽  
Large Numbers ◽  
Intracytoplasmic Membranes

The frequency of intracytoplasmic membranes in several methanogens grown on H2–CO2 varied with the conditions of growth and varied from one strain to another. Methanobacterium thermoautotrophicum often generated large numbers of intracytoplasmic membranes, while Methanospirillum hungatei produced these membranes only rarely. Conditions allowing for rapid growth, including optimal temperature and high agitation rates, increased the production of intracytoplasmic membranes. These membranes consisted mainly of vesicles composed of one or several membrane layers, often positioned in the central region of the cytoplasm. Several mesophilic methanogens could be grown such that intracytoplasmic membranes were rarely or never observed in thin section or in replicas of cross-fractures from frozen cells. Since high rates of methane synthesis still occurred in these cultures, it follows that the intracytoplasmic membrane system is not a necessary organelle for methane formation in these strains. Negative staining for electron microscopy is not an accurate method to visualize intracytoplasmic membranes in these bacterial cells.

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