Purification and characterization of an inducible dissimilatory type sulfite reductase from Clostridium pasteurianum

An inducible sulfite reductase was purified from Clostridium pasteurianum. The pH optimum of the enzyme is 7.5 in phosphate buffer. The molecular weight of the reductase was determined to be 83,600 from sodium dodecyl sulfate gel electrophoresis with a proposed molecular structure: ₂₂. Its absorption spectrum showed a maximum at 275 nm, a broad shoulder at 370 nm and a very small absorption maximum at 585 nm. No siroheme chromophore was isolated from this reductase. The enzyme could reduced the following substrates in preferential order: NH₂OH> SeO ₃ ^2- >NO ₂ ^2- at rates 50% or less of its preferred substrate SO ₃ ^2- . The proposed dissimilatory intermediates, S₃O ₆ ^2- or S₂O ₃ ^2- , were not utilized by this reductase while KCN inhibited its activity. Varying the substrate concentration [SO ₃ ^2- ] from 1 to 2.5 mol affected the stoichiometry of the enzyme reaction by alteration of the ratio of H₂ uptake to S^2- formed from 2.5:1 to 3.1:1. The inducible sulfite reductase was found to be linked to ferredoxin which could be completely replaced by methyl viologen or partially by benzyl viologen. Some of the above-mentioned enzyme properties and physiological considerations indicated that it was a dissimilatory type sulfite reductase.Abbreviations SDS sodium dodecyl sulfate - BSA bovine serum albumin - LDH Lactate dehydrogenase     

Siroheme sulfite reductase isolated from Chromatium vinosum. Purification and investigation of some of its molecular and catalytic properties

Cells of the phototrophic bacterium Chromatium vinosum strain D were shown to contain a siroheme sulfite reductase after autotrophic growth in a sulfide/bicarbonate medium. The enzyme could not be detected in cells grown heterotrophically in a malate/sulfate medium. Siroheme sulfite reductase was isolated from autotrophic cells and obtained in an about 80% pure preparation which was used to investigate some molecular and catalytic properties of the enzyme. It was shown to consist of two different types of subunits with molecular weights of 37,000 and 42,000, most probably arranged in an ₄₄-structure. The molecular weight of the native enzyme was determined to 280,000, 51 atoms of iron and 47 atoms of acid-labile sulfur were found per enzyme molecule. The absorption spectrum indicated siroheme as prosthetic group; it had maxima at 280 nm, 392 nm, 595 nm, and 724 nm. The molar extinction coefficients were determined as 302×10³ cm²xmmol^-1 at 392 nm, 98×10³ cm² xmmol^-1 at 595 nm and 22×10³ cm²x-mmol^-1 at 724 nm. With reduced viologen dyes as electron donor the enzyme reduced sulfite to sulfide, thiosulfate, and trithionate. The turnover number with 59 (2 e^-/enzyme moleculexmin) was low. The pH-optimum was at 6.0. C. vinosum sulfite reductase closely resembled the corresponding enzyme from Thiobacillus denitrificans and also desulfoviridin, the dismilatory sulfite reductase from Desulfovibrio species. It is proposed that C. vinosum catalyses anaerobic oxidation of sulfide and/or elemental sulfur to sulfite in the course of dissimilatory oxidation of reduced sulfur compounds to sulfate.Non-common abbreviations APS adenylyl sulfate - SDS sodium dodecyl sulfate     

Formation of thiosulfate and trithionate during sulfite reduction by washed cells of Desulfovibrio desulfuricans

Deenergized cells of Desulfovibrio desulfuricans strain Essex 6 formed trithionate and thiosulfate during reduction of sulfite with H₂ or formate. The required conditions were pretreatment with the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), low concentration of the electron donor H₂ or formate (25–200 M) and the presence of sulfite in excess (>250 M). The cells formed up to 20 M thiosulfate, and variable amounts of trithionate (0–9 M) and sulfide (0–62 M). Tetrathionate was not produced. Sulfate could not replace sulfite in these experiments, as deenergized cells cannot activate sulfate. However, up to 5 M thiosulfate was produced by cells growing with H₂ and excess sulfate in a chemostat. Micromolar concentrations of trithionate were incompletely reduced to thiosulfate and sulfide by washed cells in the presence of CCCP. Millimolar trithionate concentrations blocked the formation of sulfide, even in the absence of CCCP, and caused thiosulfate accumulation; sulfide formation from sulfate, sulfite or thiosulfate was stopped, too. Trithionate reduction with H₂ in the presence of thiocyanate was coupled to respiration-driven proton translocation (extrapolated H⁺/H₂ ratios of 1.5±0.6). Up to 150 M trithionate was formed by washed cells during oxidation of sulfite plus thiosulfate with ferricyanide as electron acceptor (reversed trithionate reductase activity). Cell breakage resulted in drastic decrease of sulfide formation. Cell-free extract reduced sulfite incompletely to trithionate, thiosulfate, and sulfide. Thiosulfate was reduced stoichiometrically to sulfite and sulfide (thiosulfate reductase activity). The formation of sulfide from sulfite, thiosulfate or trithionate by cell-free extract was blocked by methyl viologen, leading to increased production of thiosulfate plus trithionate from sulfite, or increased thiosulfate formation from trithionate. Our study demonstrates for the first time the formation of intermediates during sulfite reduction with whole cells of a sulfate-reducing bacterium oxidizing physiological electron donors. All results are in accordance with the trithionate pathway of sulfite reduction.With gratitude dedicated to Prof. Dr. Norbert Pfennig on occasion of his 65th birthday     

Uptake of sulfate,sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans

pH changes and sulfide production upon addition of sulfate, sulfite or thiosulfate to non-buffered H₂-saturated cell suspensions of Desulfovibrio desulfuricans were studied by means of electrodes. The addition of these electron acceptors resulted in a rapid alkalinization of the suspension which was accompanied by sulfide production. At-2° C, alkalinization without immediate sulfide production could be obtained. After addition of ³⁵S-labelled sulfate at-2° C, the label was found to be concentrated 7,500-fold in the cells, while 2 protons per sulfate molecule had disappeared from the outer bulk phase. Alkalinization and sulfide production from micromolar electron acceptor additions depended on the transmembraneous proton gradient ( pH), and were reversibly inhibited in alkaline solution (pH>8.0) or by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Protonophore-inhibited sulfide production from sulfite or thiosulfate could be restored if the cell membranes were permeabilized by the detergent cetyltrimethylammonium bromide (CTAB), or if downhill transport was made possible by the addition of electron acceptors at millimolar concentrations. Sulfate was not reduced under these conditions, presumably because the cells did not contain ATP for its activation. K⁺-and Na⁺-ionophores such as nigericin, valinomycin or monensin appeared to be of limited efficiency in D. desulfuricans. In most experiments, sulfate reduction was inhibited by the K⁺–H⁺ antiporter nigericin in the presence of K⁺, but not by the thiocyanate anion or the K⁺-transporter valinomycin. The results indicate that sulfate, sulfite and thiosulfate are taken up by proton-anion symport, presumably as undissociated acids with an electroneutral mechanism, driven by the transmembraneous pH gradient ( pH) or by a solute gradient. Kinetics of alkalinization and sulfide production in cells grown with different electron acceptors revealed that D. desulfuricans has different specific uptake systems for sulfate and thiosulfate, and obviously also for sulfite. It is proposed that the electron acceptor transport finally will not consume net energy during growth in buffered medium: The protons taken up during active electron acceptor transport leave the cell with the reduced end-product by simple passive diffusion of H₂S.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - CTAB cethyltrimethylammonium bromide     

A study on electron transport-driven proton translocation in Desulfovibrio desulfuricans

Proton translocation by washed cells of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain Essex 6 was studied by means of pH and sulfide electrodes. Reversible extrusion of protons could be induced either by addition of electron acceptors to cells incubated under hydrogen, or by addition of hydrogen to cells incubated in the presence of an appropriate electron acceptor. Proton translocation was increased in the presence of ionophores that dissipate the membrane potential (thiocyanate, methyl triphenylphosphonium cation, but not valinomycin) and was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). Upon micromolar additions of H₂, usually sulfide was formed in stoichiometric amounts, and extrapolated H⁺/H₂ ratios were 1.8±0.5 with sulfate, 2.3±0.3 with sulfite and 0.5±0.1 with thiosulfate. In several experiments hydrogen pulses caused increased proton extrusion not associated with sulfide production. This was a hint that sulfite might be reduced via intermediates. In the absence of H₂S formation, extrapolated H⁺/H₂ ratios were 3.1±0.8 with sulfate, 3.4±1.1 with sulfite, 4.4±0.8 with thiosulfate and 6.3±1.2 with oxygen. Micromolar pulses of electron acceptors to cells incubated under H₂ caused less proton translocation than H₂ pulses in presence of excess of electron acceptor; extrapolated H⁺/H₂ ratios were 1.3±0.4 with sulfite, 3.3±0.9 with nitrite and 4.2±0.5 with oxygen. No proton translocation was observed after micromolar pulses of sulfate, thiosulfate or nitrate to cells incubated under hydrogen in the presence of thiocyanate. Inhibition experiments with CO and CuCl₂ revealed that the hydrogenase activity was localized in the intracellular space, and that no periplasmic hydrogenase was present. The results indicate that D. desulfuricans can generate a proton gradient by pumping protons across the cytoplasmic membrane.Abbreviations APS adenosine 5-phosphosulfate - CCCP carbonyl cyanide m-chlorophenylhydrazone - MTTP⁺ methyl triphenylphosphonium cation     

An iron tetrahydroporphyrin prosthetic group common to both assimilatory and dissimilatory sulfite reductases

The heme² chromophore of the “assimilatory” E. coli sulfite reductase is an iron-octacarboxylic tetrahydroporphyrin of the isobacteriochlorin type (1). Although the two “dissimilatory” sulfite reductases, desulfoviridin and desulforubidin, from the sulfate reducing bacteria Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain), have absorption spectra and reaction products which differ from those of E. coli sulfite reductase, the present studies indicate that they contain prosthetic groups with an organic structure closely similar or identical to that of the E. coli sulfite reductase heme. EPR spectra show high-spin ferriheme in all three enzymes. It is clear, however, that the prosthetic groups must reside in substantially different environments within their respective proteins.     

Vectorial electron transport in Degulfovibrio vulgaris (Marburg) growing on hydrogen plus sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source in a medium containing excess iron. The topography of electron transport components was investigated. The bacterium contained per mg cells (dry weight) 30U hydrogenase (1U=1 mol/min), 35 g desulfoviridin (= bisulfite reductase), 0.6 U adenosine phosphosulfate reductase, 30 mU thiosulfate reductase, 0.3 nmol cytochrome c ₃ (M _r=13,000), 0.04 nmol cytochrome b, 0.85 nmol menaquinone, and 0.4 nmol ferredoxin. Hydrogenase (>95%) and cytochrome c ₃ (82%) were localized on the periplasmic side and desulfoviridin (95%), adenosine phosphosulfate reductase (87%), thiosulfate reductase (74%), and ferredoxin (71%) on the cytoplasmic side of the cytoplasmic membrane; menaquinone and cytochrome b were exlusively found in the membrane fraction. The location of the oxidoreductases indicate that in D. vulgaris (Marburg) H₂ oxidation and sulfate reduction take place on opposite sides of the cytoplasmic membrane rather than on the same side, as has recently been proposed.     

Sulfate formation via ATP sulfurylase in thiosulfate- and sulfite-disproportionating bacteria

Disproportionation of thiosulfate or sulfite to sulfate plus sulfide was found in several sulfate-reducing bacteria. Out of nineteen strains tested, eight disproportionated thiosulfate, and four sulfite. Growth with thiosulfate or sulfite as the sole energy source was obtained with three strains (Desulfovibrio sulfodismutans and the strains Bra02 and NTA3); additionally, D. desulfuricans strain CSN grew with sulfite but not with thiosulfate, although thiosulfate was disproportionated. Two sulfur-reducing bacteria, four phototrophic sulfur-oxidizing bacteria (incubated in the dark), and Thiobacillus denitrificans did not disproportionate thiosulfate or sulfite. Desulfovibrio sulfodismutans and D. desulfuricans CSN formed sulfate from thiosulfate or sulfite even when simultaneously oxidizing hydrogen or ethanol, or in the presence of 50 mM sulfate. The capacities of sulfate reduction and of thiosulfate and sulfite disproportionation were constitutively present. Enzyme activities required for sulfate reduction (ATP sulfurylase, pyrophosphatase, APS reductase, sulfite reductase, thiosulfate reductase, as well as adenylate kinase and hydrogenase) were detected in sufficient activities to account for the growth rates observed. ADP sulfurylase and sulfite oxidoreductase activities were not detected. Disproportionation was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) but not to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). It is proposed that during thiosulfate and sulfite disproportionation sulfate is formed via APS reductase and ATP sulfurylase, but not by sulfite oxidoreductase. Reversed electron transport must be assumed to explain the reduction of thiosulfate and sulfite by the electrons derived from APS reductase.Abbreviations CCCP Carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - APS adenosine 5-phosphosulfate (adenylylsulfate)     

Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two -, - and -subunits and that it contained the nickel porphinoid coenzyme F₄₃₀ as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the -subunit was determined. A comparison with the N-terminal sequences of the -subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity.Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO₂ (apparent V_max at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N ⁵,N¹⁰-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (H₂-forming), 33 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin reductase (coenzyme F₄₂₀ dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F₄₂₀-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent K_m values for these enzymes and the effect of salts on their activities were determined.The coenzyme F₄₂₀ present in M. kandleri was identified as coenzyme F₄₂₀-2 with 2 -glutamyl residues.Abbreviations H–S-CoM coenzyme M - CH₃–S-CoM methylcoenzyme M - H–S-HTP 7-mercaptoheptanoylthreonine phosphate - MFR methanofuran - CHO-MFR formyl-MFR - H₄MPT tetrahydromethanopterin - CHO–H₄MPT N ⁵-formyl-H₄MPT - CH=H₄MPT⁺ N ⁵,N¹⁰-methenyl-H₄MPT - CH₂=H₄MPT N ⁵,N¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - 1 U= 1 mol/min     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

A light-enhanced metabolism of sulfite in cells of Cucumis sativus L. cotyledons

The effects of light and several photosynthetic inhibitors on the rate of sulfite metabolism in cells obtained from Cucumis sativus L. cotyledons was studied. The cells were treated with 200 M Na₂SO₃ and the disappearance of sulfite was monitored using either dithiobisnitrobenzoic acid or fuchsin. The rate of sulfite disappearance in light was double the dark rate. Disalicylidene propanediamine at 1 mM increased this light-enhanced metabolism approx. 50%; neither 1 M 3,4-dichlorophenyl-N,N-dimethylurea nor 0.1 mM cyanazine, which completely inhibited CO₂-dependent oxygen evolution, affected the rate of sulfite metabolism. Addition of 200 M Na₂SO₃ to the cells partially inhibited ¹⁴CO₂ fixation. The rate of sulfite consumption by the cells did not affect this inhibition. We conclude that light-dependent sulfite metabolism is cucumber cells may utilize reduced ferredoxin generated as a result of photosynthetic electron transport. An injurious interaction between CO₂ fixation and sulfite appears to occur independently of the sulfite-metabolism process.Abbreviations DCMU 3,4-dichlorophenyl-N,N-dimethylurea - DSPD disalicylidene propanediamine - DTNB 5,5-dithiobis-(2-nitrobenzoic acid)     

Regulation and function of ferredoxin-linked versus cytochrome b-linked hydrogenase in electron transfer and energy metabolism of Methanosarcina barkeri MS

Acetate-grown cells of Methanosarcina barkeri MS were found to form methane from H₂:CO₂ at the same rate as hydrogen-grown cells. Cells grown on acetate had similar levels of soluble F₄₂₀-reactive hydrogenase I, and higher levels of cytochrome-linked hydrogenase II compared to hydrogen-grown cells. The hydrogenase I and II activities in the crude extract of acetate-grown cells were separated by differential binding properties to an immobilized Cu²⁺ column. Hydrogenase II did not react with ferredoxin or F₄₂₀, whereas hydrogenase I coupled to both ferredoxin and F₄₂₀. A reconstituted soluble protein system composed of purified CO dehydrogenase, F₄₂₀-reactive hydrogenase I fraction, and ferredoxin produced H₂ from CO oxidation at a rate of 2.5 nmol/min · mg protein. Membrane-bound hydrogenase II coupled H₂ consumption to the reduction of CoM-S-S-HTP and the synthesis of ATP. The differential function of hydrogenase I and II is ascribed to ferredoxin-linked hydrogen production from CO and cytochrome b-linked H₂ consumption coupled to methanogenesis and ATP synthesis, respectively.     

Sulfite: Preferential sulfur source and modifier of CO2 fixation in Chlorella vulgaris

Sulfite was added at the time of inoculation to a standard and to a sulfate deficient medium of Chlorella vulgaris. It was not only used as a sulfur source, but besides this, at concentrations <1.0 mmol l^–1, the growth yield was enhanced up to 30% compared to sulfate saturated conditions. Higher sulfite concentrations increasingly inhibited cell growth. Growth rate determinations indicated that the enhancement, and the inhibition respectively, were confined to the very beginning of culture growth; the time period during which the sulfite was not yet oxidized (5–10 h). In contrast, an increased CO₂ fixation rate/unit of protein, occurring up to 5.0 mmol l^–1 sulfite and a shift towards the -carboxylation pathway, are persisting at least during the growth period of 4 days. The preferential uptake of sulfite, also indicated by a marked increase in methionine content of algal protein, presumably causes an increase in thylakoidal sulfolipids, and is such modifying the CO₂ fixation.Abbreviations PGA 3-phosphoglyceric acid - APS adenosine 5-phosphosulfate - PEP phosphoenolpyruvate     

Expression, purification and characterization of the sulfite reductase hemo-subunit, SiR-HP, from Acidithiobacillus ferrooxidans

Sulfite reductase (SiR) is a large and soluble enzyme which catalyzes the transfer of six electrons from NADPH to sulfite to produce sulfide. The sulfite reductase flavoprotein (SiR-FP) contains both FAD and FMN, and the sulfite reductase hemoprotein (SiR-HP) contains an iron-sulfur cluster coupled to a siroheme. The enzyme is arranged so that the redox cofactors in the FAD-FMN-Fe(4)S(4)-Heme sequence make an electron pathway between NADPH and sulfite. Here we report the cloning, expression, and characterization of the SiR-HP of the sulfite reductase from Acidithiobacillus ferrooxidans. The purified SiR-HP contained a [Fe(4)S(4)] cluster. Site-directed mutagenesis results revealed that Cys427, Cys433, Cys472 and Cys476 were in ligating with the [Fe(4)S(4)] cluster of the protein.     

N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus

Methylene-H₄MPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F₄₂₀ as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a K _m for methylene-H₄MPT of 16 M, a K _m for F₄₂₀H₂ of 4 M, and a V _max of 450 U/mg (K_cat=265 s^-1) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F₄₂₀ (0.2 mM), methylene-H₄MPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-H₄MPT reductase (F₄₂₀-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described.Abbreviations H₄MPT tetrahydromethanopterin - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - CHH₄MPT methenyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formyl-MFR - 1 U 1 mol/min     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Purification and Characterization of Membrane-Associated Hydrogenase from the Deep-Sea Epsilonproteobacterium Hydrogenimonas thermophila

Membrane-associated hydrogenase was purified from the chemolithoautotrophic epsilonproteobacterium Hydrogenimonas thermophila at 152-fold purity. The hydrogenase was found to be localized in the periplasmic space, and was easily solubilized with 0.1% Triton X-100 treatment. Hydrogen oxidation activity was 1,365 μmol H₂/min/mg of protein at 80 °C at pH 9.0, with phenazine methosulphate as the electron acceptor. Hydrogen production activity was 900 μmol H₂/min/mg of protein at 80 °C and pH 6.0, with reduced methyl viologen as the electron donor. The hydrogenase from this organism showed higher oxygen tolerance than those from other microorganisms showing hydrogen oxidation activity. The structural genes of this hydrogenase, which contains N-terminal amino acid sequences from both small and large subunits of purified hydrogenase, were successfully elucidated. The hydrogenase from H. thermophila was found to be phylogenetically related with H₂ uptake hydrogenases from pathogenic Epsilonproteobacteria.     

Properties of an enzymatic complex active in sulfite and thiosulfate oxidation by Rhodotorula sp.

An enzymatic complex from Rhodotorula was characterized and it was indicated that it possessed thiosulfate-oxidizing activity, forming tetrathionate as well as sulfite oxidase activity. Both activities coupled with ferricyanide and native cytochrome c but no with mammalian cytochrome c. Activities of these enzymes were inhibited by thiol inhibitors. Chelating agents did not affect thiosulfate oxidizing activity and only moderately inhibited sulfite oxidase. Both activities disappeared after treatment with proteolytic enzymes or sodium deoxycholate which indicates an essential role played not only by protein but also by phospholipids in the enzymatic activity of the complex. Thiosulfate oxidizing enzyme had a K _m for thiosulfate of 0.16 mM with ferricyanide as electron acceptor and of 14 M with native cytochrome c and of 0.34 mM for ferricyanide. Optimum pH for this activity was 7.8. Other properties of this enzyme were similar to those of thiobacilli and heterotrophic bacteria. The activity of sulfite oxidase was inhibited by 50% with 10 M AMP. The K _m values of this enzyme were 1 mM with ferricyanide as electron acceptor and 60 M with native cytochrome c for sulfite and 0.42 mM for ferricyanide. The enzyme did not show a specific optimum pH value with ferricyanide as electron acceptor. However, with native cytochrome c optimum pH was 7.8 for its activity. In many properties the sulfite oxidase from Rhodotorula was similar to the enzyme from Thiobacillus ferrooxidans, T. concretivorus, T. thioparus and T. novellus.Abbreviations CSH reduced glutathion - APS reductase, adenosine-S-phosphosulfate reductase - pHMB p-hydroxymercuribenzoate - NEM N-ethylmalcimide - TCA trichloroacetic acid - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen-bis 5-phenyloxazol     

The soluble [NiFe]-hydrogenase from<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> contains four cyanides in its active site,one of which is responsible for the insensitivity towards oxygen

Infrared spectra of ¹⁵N-enriched preparations of the soluble cytoplasmic NAD⁺-reducing [NiFe]-hydrogenase from Ralstonia eutropha are presented. These spectra, together with chemical analyses, show that the Ni-Fe active site contains four cyanide groups and one carbon monoxide molecule. It is proposed that the active site is a (RS)₂(CN)Ni(-RS)₂Fe(CN)₃(CO) centre (R=Cys) and that H₂ activation solely takes place on nickel. One of the two FMN groups (FMN-a) in the enzyme can be reversibly released upon reduction of the enzyme. It is now reported that at longer times also one of the cyanide groups, the one proposed to be bound to the nickel atom, could be removed from the enzyme. This process was irreversible and induced the inhibition of the enzyme activity by oxygen; the enzyme remained insensitive to carbon monoxide. The Ni-Fe active site was EPR undetectable under all conditions tested. It is concluded that the Ni-bound cyanide group is responsible for the oxygen insensitivity of the enzyme.Abbreviations BV benzyl viologen - DCIP 2,6-dichlorophenol-indophenol - EXAFS extended X-ray absorption fine structure - FTIR Fourier transform infrared - MV methyl viologen - SH soluble NAD⁺-reducing hydrogenase - XAS X-ray absorption spectroscopy     

Chemolithotrophic growth ofDesulfovibrio desulfuricans with hydrogen coupled to ammonification of nitrate or nitrite

Two of nine sulfate reducing bacteria tested,Desulfobulbus propionicus andDesulfovibrio desulfuricans (strain Essex 6), were able to grow with nitrate as terminal electron acceptor, which was reduced to ammonia. Desulfovibrio desulfuricans was grown in chemostat culture with hydrogen plus limiting concentrations of nitrate, nitrite or sulfate as sole energy source. Growth yields up to 13.1, 8.8 or 9.7 g cell dry mass were obtained per mol nitrate, nitrite or sulfate reduced, respectively. The apparent half saturation constants (K _s) were below the detection limits of 200, 3 or 100 mol/l for nitrate, nitrite of sulfate, respectively. The maximum growth rates {ie63-1} raised from 0.124 h^-1 with sulfate and 0.150 h^-1 with nitrate to 0.193 h^-1 with nitrite as electron acceptor. Regardless of the electron acceptor in the culture medium, cell extracts exhibited absorption maxima corresponding to cytochromec and desulfoviridin. Nitrate reductase was found to be inducible by nitrate or nitrite, whereas nitrite reductase was synthesized constitutively. The activities of nitrate and nitrite reductases with hydrogen as electron donor were 0.2 and 0.3 mol/min·mg protein, respectively. If limiting amounts of hydrogen were added to culture bottles with nitrate as electron acceptor, part of the nitrate was only reduced to the level of nitrite. In media containing nitrate plus sulfate or nitrite plus sulfate, sulfate reduction was suppressed.The results demonstrate that the ammonification of nitrate or nitrite can function as sole energy conserving process in some sulfate-reducing bacteria.