Metabolism of sulfate-reducing prokaryotes

Dissimilatory sulfate reduction is carried out by a heterogeneous group of bacteria and archaea that occur in environments with temperatures up to 105 °C. As a group together they have the capacity to metabolize a wide variety of compounds ranging from hydrogen via typical organic fermentation products to hexadecane, toluene, and several types of substituted aromatics. Without exception all sulfate reducers activate sulfate to APS; the natural electron donor(s) for the ensuing APS reductase reaction is not known. The same is true for the reduction of the product bisulfite; in addition there is still some uncertainty as to whether the pathway to sulfide is a direct six-electron reduction of bisulfite or whether it involves trithionate and thiosulfate as intermediates. The study of the degradation pathways of organic substrates by sulfate-reducing prokaryotes has led to the discovery of novel non-cyclic pathways for the oxidation of the acetyl moiety of acetyl-CoA to CO₂. The most detailed knowledge is available on the metabolism ofDesulfovibrio strains, both on the pathways and enzymes involved in substrate degradation and on electron transfer components and terminal reductases. Problems encountered in elucidating the flow of reducing equivalents and energy transduction are the cytoplasmic localization of the terminal reductases and uncertainties about the electron donors for the reactions catalyzed by these enzymes. New developments in the study of the metabolism of sulfate-reducing bacteria and archaea are reviewed.     

Immunocytochemical localization of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum

Cells of Methanobacterium thermoautotrophicum were fixed with glutaraldehyde, sectioned and labeled with antibodies against the subunit of component C (=methyl-CoM reductase) of methyl-CoM reductase system and with colloidal gold-labeled protein A. It was found that the gold particles were located predominantly in the vicinity of the cytoplasmic membrane, when the cells were grown under conditions where methyl-CoM reductase was not overproduced. This finding confirms the recent data obtained with Methanococcus voltae showing via the same immunocytochemical localization technique that in this organism methyl-CoM reductase is membrane associated.     

Import of peroxisomal hydroxypyruvate reductase into glyoxysomes

A new procedure was used to purify the peroxisomal matrix enzyme hydroxypyruvate reductase (HPR) from green leaves of pumpkin (Cucurbita pepo L.) and spinach (Spinacia oleracea L.). Monospecific antibodies were prepared against this enzyme in rabbits. Immunoprecipitation of HPR from watermelon (Citrullus vulgaris Schrad.) yielded a single protein with a subunit molecular weight of 45 kDa. Immunohistochemical labeling of HPR was found exclusively in watermelon microbodies. Isolated polyadenylated mRNA from light-grown watermelon cotyledons was injected into Xenopus laevis oocytes. The heterologous in-vivo translation product of HPR exhibited the same molecular weight as the immunoprecipitate from watermelon cotyledons, indicating the lack of a cleavable extra sequence. The watermelon HPR translated in oocytes was imported into isolated glyoxysomes from castor bean (Ricinus communis L.) endosperm and remained resistant to proteolysis after the addition of proteinase K. The HPR did not change its apparent molecular weight during sequestration; however, it may have changed its conformation.Abbreviations HPR hydroxypyruvate reductase - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Periplasmic location of nitrous oxide reductase and its apoform in denitrifying Pseudomonas stutzeri

Immunogold labelling techniques on ultrathin sections of low temperature embedded cells yielded evidence for the periplasmic location of the respiratory enzymes N₂O reductase and nitrite reductase (cytochrome cd ₁) in Pseudomonas stutzeri strain ZoBell. Cell fractionation by spheroplast preparation and two-dimensional electrophoresis showed the absence of a membrane association of these enzymes. Immunocytochemical localization of N₂O reductase in a mutant strain deficient in the chromophore of N₂O reductase showed the gold label at the cell periphery, indicating that the copper chromophore processing takes place after export of this protein's apoform.     

Proline dehydrogenase and pyrroline-5-carboxylate reductase from pumpkin cotyledons

Activity of proline dehydrogenase and pyrroline-5-carboxylate reductase was greatest after 5 and 7 days germination in green and etiolated cotyledons respectively of pumpkin (Cucurbita moschata Poir. cv. Dickinson Field). The ratio of pyrroline-5-carboxylate reductase to proline dehydrogenase activity was constant throughout germination. Both enzymes were purified 30-fold but the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase activity was constant throughout purification. However, this ratio decreased with storage, especially in purified preparations. Both enzymes were stable at high temperature and the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase remained unchanged on heating. Proline dehydrogenase and pyrroline-5-carboxylate reductase were inhibited by sodium bisulfite and cysteine. ATP, ADP and NADP caused inhibition of both enzymes. Proline dehydrogenase utilized NAD but not NADP. Pyrroline-5-carboxylate reductase had a 2.5-fold greater activity with NADH than NADPH. Most of the data presented suggest that proline dehydrogenase and pyrroline-5-carboxylate reductase activities occur on the same protein molecule.     

The effect of vanadium on nitrate reductase of Chlorella vulgaris

Added vanadate ions inhibit purified nitrate reductase from Chlorella vulgaris by reacting with the enzyme in a manner rather similar to that of HCN. Thus vanadate, like HCN, forms an inactive complex with the reduced enzyme, and this inactivated enzyme can be reactivated rapidly by adding ferricyanide. The inactive vanadate enzyme complex is less stable than the inactive HCN complex, and the two can be distinguished by the fact that EDTA causes a partial reactivation of the former, but not of the latter. Vanadate can also cause an increase in HCN formation by intact Chlorella vulgaris cells. When these cells were incubated with vanadate, their nitrate reductase was reversibly inactivated, and all of this inactive enzyme could be shown to be the HCN complex rather than the vanadate complex. When HCN and vanadate are both present, the HCN-inactivated enzyme, being more stable, will be formed in preference to the vanadate-inactivated enzyme.Abbreviation EDTA ethylenediamine tetraacetate     

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.     

Characterization of the membranous denitrification enzymes nitrite reductase (cytochrome cd1) and copper-containing nitrous oxide reductase from Thiobacillus denitrificans

Cytochrome cd ₁-nitrite reductase and nitrous oxide reductase of Thiobacillus denitrificans were purified and characterized by biochemical and immunochemical methods. In contrast to the generally soluble nature of the denitrification enzymes, these two enzymes were isolated from the membrane fraction of T. denitrificans and remained active after solubilization with Triton X-100. The properties of the membrane-derived enzymes were similar to those of their soluble counterparts from the same organism. Nitrous oxide reductase activity was inhibited by acetylene. Nitrite reductase and nitrous oxide reductase cross-reacted with antisera raised against the soluble enzymes from Pseudomonas stutzeri. The nirS, norBC, and nosZ genes encoding the cytochrome cd ₁-nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively, from P. stutzeri hybridized with genomic DNA from T. denitrificans. Cross-reactivity and similar N-terminal amino acid and gene sequences suggest that the primary structures of the Thiobacillus enzymes are homologous to the soluble proteins from P. stutzeri. Received: 18 August 1995 / Accepted: 30 October 1995     

Thermotolerance of adenylylsulfate reductase from Thiobacillus denitrificans

Abstract Adenylylsulfate (APS) and APS reductase are important in the energy-generating processes of sulfate-reducing bacteria and sulfur lithotrophs (phototrophs and nonphototrophs). APS reductase from an extremely thermophilic archaebacterial sulfate-reducer was recently shown to be thermophilic with optimal activity at 85°C (Speich and Truper (1988) J. Gen. Microbiol. 134, 1419–1425). APS reductase of Thiobacillus denitrificans , a mesophilic eubacterium, has biochemical and physical properties in common with the thermophilic enzyme and is also thermotolerant (up to 75°C). APS reductase and other enzymes of dissimilative inorganic sulfur metabolism may commonly be thermotolerant is mesophilic eubacteria; perhaps a vestige of their primordial significance.     

The correlation between the protein composition of cytoplasmic membranes and the formation of nitrate reductase A,chlorate reductase C and tetrathionate reductase in Proteus mirabilis wild type and some chlorate resistant mutants

Three genotypically different chlorate resistant mutants, chl I, chl II and chl III, appeared to lack completely nitrate reductase A, chlorate reductase C and tetrathionate reductase activity. Fumarate reductase is only partially affected in chl I and chl III and unaffected in chl II. Formate dehydrogenase is only partially diminished in chl II, hydrogenase is diminished in chl I and chl II and completely absent in chl III.Subunits of nitrate reductase A, chlorate reductase C and tetrathionate reductase have been identified in protein profiles of purified cytoplasmic membranes from the wild type and the three mutant strains, grown under various conditions. Only the presence and absence of the largest subunits of these enzymes appeared to be correlated with their repression and derepression in the wild type membranes. On the cytoplasmic membranes of the chl I and chl III mutants these subunits lack for the greater part. In the chl II mutant, however, these subunits are inserted in the membrane all together after anaerobic growth with or without nitrate.A model for the repression/derepression mechanism for the reductases has been proposed. It includes repression by cytochrome b components, whereas the redox-state of the nitrate reductase A molecule itself is also involved in its derepression under anaerobic conditions.     

Nitrite activation of nitrate reductase in higher plants

Comparative studies of nitrate-activated nitrate reductase (NR-NO₂) and nitrate-induced nitrate reductase (NR-NO₃) (EC 1.6.6.2) indicate that the enzymes differ in structure, heat stability, and pH dependence, but have the same cofactor requirment. NR-NO₂ developes in barley (Hordeum vulgare L. var. Dvir) seedlings as NR-NO₃ disappears. A transition from the active to the inactive form of nitrate reductase takes place. Nitrite seems to activate the inactive form of the enzyme.     

Activity of nitrate reductase in <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> VKM 1388

Evidence was obtained of the inhibitory effect of nitrate on the metabolism of Desulfovibrio vulgaris 1388. Nitrate is reduced only at low concentrations and in the presence of sulfate in the medium. Genetic data suggest that the genome of D. vulgaris 1388 contains the information about the γ subunit and possibly the NarG catalytic subunit of the membrane-bound nitrate reductase.     

Screening of monoclonal antibodies to scarce and labile enzymes: A functional immunoassay for isolating monoclonal antibodies to NADPH:nitrate reductase

A functional immunoassay, that has proved very useful, is described for screening and identifying monoclonal antibodies (McAbs) against scarce and labile enzymes. This method does not require purified enzyme or antigen and it has been successfully applied to isolate three hybridomas secreting McAbs to NADPH:nitrate reductase from the chloronema cells of the mossFunaria hygrometrica. Briefly, the protocol involves: adsorption of murine antibodies from hybridoma supernatants by rabbit antimouse IgG antibody pre-adsorbed toStaphylococcus aureus cells (SAC), reaction with crude extract for 15 min, sedimentation of the SAC complex by centrifugation and measurement of residual enzymatic activity in the supernatant. A depletion indicates the presence of antibodies that bind to the active enzyme. The method is rapid, sensitive and versatile enough to be used to isolate McAbs with exquisite specificities. The three isolated McAbs recognized nitrate reductase protein in a conformation-independent and/or a conformation-dependent manner.     

The light-induced development of nitrate reductase in etiolated barley shoots: An inhibitory effect of laevulinic acid

Induction of nitrate reductase EC 1.6.6.1 in etiolated barley (Hordeum vulgare L., var. Proctor) required continuous illumination and showed a lag period of about three hours. During the first 16 h of illumination the ratio NADH/NAD and NADPH/NADP, taken as a measure of internal oxidation reduction potential, declined. The inhibitor DCMU applied to whole leaves at concentrations shown to inhibit the reduction of cytochrome f by Photosystem 2 light did not inhibit the induction of nitrate reductase nor did it diminish the ratio of reduced to oxidised puridine nucleotides in the early hours of greening. It was concluded that light driven electron flow was not necessary for nitrate reductase induction. Chloramphenicol gave a slight inhibition of nitrate reductase induction. Laevulinic acid was added to greening barley leaves to inhibit tetrapyrrole pigment biosynthesis and plastid development. It strongly inhibited chlorophyll synthesis and nitrate reductase induction, with relatively little effect upon Photosystem 1 and 2 activities in isolated plastids. The activities of other inducible enzymes and control enzymes were little affected by laevulinic acid. Laevulinic acid also inhibited nitrate reductase induction by added nitrate in fully-greened illuminated plants grown in nitrate-free medium and so is unlikely to be acting through inhibition of plastid development. This inhibitor lowered the level of protohaem in whole leaves and plastids of greening barley and it is postulated that it may diminish the protohaem available for the assembly of a cytochrome b component of nitrate reductase.Abbreviations DCMU 3-(3:4-Dichlorophenyl)-1:1-dimethylurea - LA laevulinic acid     

In-situ localization of nitrate reductase in maize roots

The localization of nitrate reductase (NR; EC 1.6.6.2) in cells of root tissues ofZea mays L. (W64A W182L) was determined using post-embedding immunogold labeling at the electron-microscopy level and using silver enhancement of the colloidal-gold signal for light microscopy. Nitrate reductase is located in the cytoplasm of root epidermal and cortical cells, and in the cells of the parenchyma and pericycle within the vascular cylinder. A weaker signal was also obtained in parenchymal cells of the pith lying next to the xylem. A positive signal for NR protein was seen in the chloroplast fraction of maize leaves and in the plastid fraction of roots. This signal was lost when affinity-purified antibodies were used. Sections of Lowicryl-embedded tissue were found to be suitable for the localization of the non-abundant NR protein when adequate controls and signal-enhancement procedures were used.Abbreviations IgG immunoglobulin G - NR nitrate reductase - PEPCase phosphoenolpyruvate carboxylase This research was funded by Natural Sciences and Engineering Research Council (NSERC) of Canada grants ISE0125461 (AO), OGP0106265 (JSG) and an NSERC Visiting Scientist Award to E.F.     

Fumarate reductase of Clostridium formicoaceticum

When Clostridium formicoaceticum was grown on fumarate or l-malate crude cell extracts contained a high fumarate reductase activity. Using reduced methyl viologen as electron donor the specific activity amounted to 2–3.5 U per mg of protein. Reduced benzyl viologen, FMNH₂ and NADH could also serve as electron donors but the specific activities were much lower. The NADH-dependent activity was strictly membrane-bound and rather labile. Its specific activity did not exceed 0.08 U per mg of particle protein. Fumarate reductase activity was also found in cells of C. formicoaceticum grown on fructose, gluconate, glutamate and some other substrates.The methyl viologen-dependent fumarate reductase activity could almost completely be measured with intact cells whereas only about 25% of the cytoplasmic acetate kinase activity was detected with cell suspensions. The preparation of spheroplasts from cells of C. formicoaceticum in 20 mM HEPES-KOH buffer containing 0.6 M sucrose and 1 mM dithioerythritol resulted in the specific release of 88% of the fumarate reductase activity into the spheroplast medium. Only small amounts of the cytoplasmic proteins malic enzyme and acetate kinase were released during this procedure. These results indicate a peripheral location of the fumarate reductase of C. formicoaceticum on the membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - O.D optical density - DTE dithioerythritol     

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     

Mechanism of dissimilatory sulfite reduction by Desulfovibrio desulfuricans: purification of a membrane-bound sulfite reductase and coupling iwth cytochrome c 3 and hydrogenase

The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO₃ ^-H₂S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c ₃ from D. desulfuricans. The specific activity of mSiR was 103 nmol H₂ min^-1 mg^-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO₃ ^-H₂S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations SiR sulfite reductase - mSiR sulfite reductase purified from membranes - sSiR sulfite reductase purified from the soluble fraction Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c ₃ hydrogenase, EC 1.12.2.1     

A comparison of the high-active and low-active form of nitrate reductase in synchronous Chlorella sorokiniana

Chlorella sorokiniana possesses two forms of nitrate reductase (EC 1.6.6.1.). One with low activity is present in cells at the end of the light-dark cycle, the other with high activity is present after 1 h of illumination. The two forms can be distinguished by gel electrophoresis, isopycnic centrifugation, assay of the partial reactions and their sensitivity to antibodies, respectively. These differences are discussed with respect to an effect of intracellular nitrate on the activation of nitrate reductase.Abbreviations NAR nitrate reductase - FMN flavine mononucleotide - MV methylviologen     

Polysulfide reductase and formate dehydrogenase from Wolinella succinogenes contain molybdopterin guanine dinucleotide

Pterin derivatives were extracted from formate dehydrogenase and from polysulfide reductase of Wolinella succinogenes and converted to 6-carboxypterin. The amounts of 6-carboxypterin were consisted with the molybdenum content of the enzymes. The bis(carboxamidomethyl) derivatives of the cofactors showed absorption spectra that were identical with that of the corresponding molybdopterin guanine dinucleotide derivative (cam MGD). After hydrolysis of the derivatives with nucleotide pyrophosphatase in the presence of alkaline phosphatase, guanosine was formed together with a compound showing the properties of dephospho-bis(carboxamidomethyl)-molybdopterin. It is conluded that both formate dehydrogenase and polysulfide reductase of W. succinogenes contain molybdopterin guanine dinucleotide.Abbreviations MPT molybdopterin - MGD molybdopterin guanine dinucleotide - cam MPT bis(carboxyamidomethyl)-molybdopterin - cam MGD bis(carboxyamidomethyl)-molybdopterin guanine dinucleotide