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)     

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     

The existence of an alternative electron-transfer pathway to the periplasmic nitrite reductase (cytochrome cd 1) in Paracoccus denitrificans

Exposure of aerobically-grown wild-type cells of Paracoccus denitrificans to a decreased aeration caused parallel increases in both PMS/ascorbate and succinate-linked activities of nitrite reductase. By contrast, the expression of the succinate-linked activity was considerably delayed in an insertion mutant specifically lacking the periplasmic 15 kDa cytochrome c-550. In this case the observed activity followed very closely the content of a 40 kDa cytochrome c. A subcellular fraction enriched in a haemoprotein of a similar apparent molecular weight showed the activity of cytochrome c peroxidase and was able to restore the antimycin-sensitive electron transport from membrane vesicles to nitrite reductase. It is concluded that P. denitrificans possesses an alternative nitrite-reducing pathway involving the 40 kDa cytochrome c instead of cytochrome c-550. This pathway branches from the respiratory chain after the cytochrome bc ₁ segment.Abbreviations PAGE polyacrylamide gel electrophoresis - PMS phenazine methosulphate     

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.Abbreviation TMAO trimethylamine-N-oxide     

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     

Isolation,sequencing and mutational analysis of a gene cluster involved in nitrite reduction inParacoccus denitrificans

By using the gene encoding the C-terminal part of thecd ₁-type nitrite reductase ofPseudomonas stutzeri JM300 as a heterologous probe, the corresponding gene fromParacoccus denitrificans was isolated. This gene,nirS, codes for a mature protein of 63144 Da having high homology withcd ₁-type nitrite reductases from other bacteria. Directly downstream fromnirS, three othernir genes were found in the ordernirECF. The organization of thenir gene cluster inPa. denitrificans is different from the organization ofnir clusters in some Pseudomonads.nirE has high homology with a S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (uro'gen III methylase). This methylase is most likely involved in the hemed ₁ biosynthesis inPa. denitrificans. The third gene,nirC, codes for a small cytochromec of 9.3 kDa having high homology with cytochromec _55X ofPs. stutzeri ZoBell. The 4th gene,nirF, has no homology with other genes in the sequence databases and has no relevant motifs. Inactivation of either of these 4 genes resulted in the loss of nitrite and nitric oxide reductase activities but not of nitrous oxide reductase activity.nirS mutants lack thecd ₁-type nitrite reductase whilenirE, nirC andnirF mutants produce a small amount ofcd ₁-type nitrite reductase, inactive due to the absence of hemed ₁. Upstream from thenirS gene the start of a gene was identified which has limited homology withnosR, a putative regulatory gene involved in nitrous oxide reduction. A potential FNR box was identified between this gene andnirS.Abbreviations SDS sodium dodecyl sulfate - NBT nitroblue tetrazolium - PAGE polyacrylamide gel electrophoresis     

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     

Formation of active nitrite reductase (cytochromecd 1) in the strainParacoccus denitrificans HUUG25

Strain HUUG25 ofParacoccus denitrificans has been frequently thought to be devoid of allc-type cytochromes. We show here by means of enzymological and immunological techniques that the mutant synthesizes active nitrite reductase (cytochromecd ₁) upon prolonged exposure to microoxic conditions. The synthesis occurred faster in the presence of exogenous hemin. The time pattern of 5-aminolevulinate synthase activity was also altered by the mutation. These findings suggest a defective regulation of heme supply to the site of nitrite reductase assembly in the periplasm.     

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

Analysis of cytoplasmic membrane from wild type and mutant Paracoccus denitrificans containing excess nitrate reductase protein and cytochrome b

Cytoplasmic membranes were isolated from wild type and mutants strain M-1 of Paracoccus denitrificans grown with low aeration to promote synthesis of nitrate reductase protein and cytochrome b. The presence of 10-100-fold excess of nitrate reductase in the wild type or the corresponding enzymically inactive protein in the mutant did not significantly affect respiratory oxidase activities with NADH, succinate or TMPD-ascorbate as electron donor. A cytochrome b-nitrate reductase complex was resolved by isoelectric focussing of Triton X-100 solubilized membranes from the wild type grown with azide and from the mutant, whereas the enzyme complex from nitrate-grown wild type was not resolved from cytochrome c. Preparations from azideinduced wild type or from the mutant could be a suitable source of the cytochrome b associated with nitrate reductase for more detailed studies.Non standard abbreviations IEF isoelectric focussing - TMPD N, N, N, N-tetramethylphenylenediamine - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide M _r =150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrane protein M _r =150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are coregulated and that the active enzyme has a role in regulating its own synthesis.Non-standard Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DOC sodlum deoxycholate     

Nitrite reductase gene from Synechococcus sp. PCC 7942: homology between cyanobacterial and higher-plant nitrite reductases

The gene encoding nitrite reductase (nir) from the cyanobacterium Synechococcus sp. PCC 7942 has been identified and sequenced. This gene comprises 1536 nucleotides and would encode a polypeptide of 56506 Da that shows similarity to nitrite reductase from higher plants and to the sulfite reductase hemoprotein from enteric bacteria. Identities found at positions corresponding to those amino acids which in the above-mentioned proteins hold the Fe₄S₄-siroheme active center suggest that nitrite reductase from Synechococcus bears an active site much alike that present in those reductases. The fact that the Synechococcus and higher-plant nitrite reductases are homologous proteins gives support to the endosymbiont theory for the origin of chloroplasts.     

Metabolic regulation including anaerobic metabolism inParacoccus denitrificans

Under anaerobic circumstances in the presence of nitrateParacoccus denitrificans is able to denitrify. The properties of the reductases involved in nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase are described. For that purpose not only the properties of the enzymes ofP. denitrificans are considered but also those fromEscherichia coli, Pseudomonas aeruginosa, andPseudomonas stutzeri. Nitrate reductase consists of three subunits: the subunit contains the molybdenum cofactor, the subunit contains the iron sulfur clusters, and the subunit is a special cytochromeb. Nitrate is reduced at the cytoplasmic side of the membrane and evidence for the presence of a nitrate-nitrite antiporter is presented. Electron flow is from ubiquinol via the specific cytochromeb to the nitrate reductase. Nitrite reductase (which is identical to cytochromecd ₁) and nitrous oxide reductase are periplasmic proteins. Nitric oxide reductase is a membrane-bound enzyme. Thebc ₁ complex is involved in electron flow to these reductases and the whole reaction takes place at the periplasmic side of the membrane. It is now firmly established that NO is an obligatory intermediate between nitrite and nitrous oxide. Nitrous oxide reductase is a multi-copper protein. A large number of genes is involved in the acquisition of molybdenum and copper, the formation of the molybdenum cofactor, and the insertion of the metals. It is estimated that at least 40 genes are involved in the process of denitrification. The control of the expression of these genes inP. denitrificans is totally unknown. As an example of such complex regulatory systems the function of thefnr, narX, andnarL gene products in the expression of nitrate reductase inE. coli is described. The control of the effects of oxygen on the reduction of nitrate, nitrite, and nitrous oxide are discussed. Oxygen inhibits reduction of nitrate by prevention of nitrate uptake in the cell. In the case of nitrite and nitrous oxide a competition between reductases and oxidases for a limited supply of electrons from primary dehydrogenases seems to play an important role. Under some circumstances NO formed from nitrite may inhibit oxidases, resulting in a redistribution of electron flow from oxygen to nitrite.P. denitrificans contains three main oxidases: cytochromeaa ₃, cytochromeo, and cytochromeco. Cytochromeo is proton translocating and receives its electrons from ubiquinol. Some properties of cytochromeco, which receives its electrons from cytochromec, are reported. The control of the formation of these various oxidases is unknown, as well as the control of electron flow in the branched respiratory chain. Schemes for aerobic and anaerobic electron transport are given. Proton translocation and charge separation during electron transport from various electron donors and by various electron transfer pathways to oxygen and nitrogenous oxide are given. The extent of energy conservation during denitrification is about 70% of that during aerobic respiration. In sulfate-limited cultures (in which proton translocation in the NADH-ubiquinone segment of the respiratory chain is lost) the extent of energy conservation is about 60% of that under substrate-limited conditions. These conclusions are in accordance with measurements of molar growth yields.     

Anaerobic oxidation of thiosulfate and elemental sulfur in Thiobacillus denitrificans

Thiobacillus denitrificans strain RT could be grown anaerobically in batch culture on thiosulfate but not on other reduced sulfur compounds like sulfide, elemental sulfur, thiocyanate, polythionates or sulfite. During growth on thiosulfate the assimilated cell sulfur was derived totally from the outer or sulfane sulfur. Thiosulfate oxidation started with a rhodanese type cleavage between sulfane and sulfone sulfur leading to elemental sulfur and sulfite. As long as thiosulfate was present elemental sulfur was transiently accumulated within the cells in a form that could be shown to be more reactive than elemental sulfur present in a hydrophilic sulfur sol, however, less reactive than sulfane sulfur of polythionates or organic and inorganic polysulfides. When thiosulfate had been completely consumed, intracellular elemental sulfur was rapidly oxidized to sulfate with a specific rate of 45 natom S°/min·mg protein. Extracellularly offered elemental sulfur was not oxidized under anaerobic conditions.     

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     

Purification and Properties of Sulfite Reductase from Desulfovibrio vulgaris,Miyazaki F

The sulfite reductase of Desulfovibrio vulgaris, strain Miyazaki F (MF), was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Ultrogel AcA34, and hydroxylapatite. The molecular weight was estimated to be 180,000 by gel filtration. It had a subunit structure of α₂β₂; the molecular weight of the α subunit was 50,000 and that of β, 39,000. The absorption spectrum with characteristic peaks at 629 and 409 nm and the amino acid composition resembled those of the sulfite reductase from D. vulgaris, Miyazaki K. The MF enzyme reduced sulfite to trithionate, thiosulfate, and sulfide by hydrogen when coupled with a hydrogenase-methyl viologen system, like other sulfite reductases from Desulfovibrio.     

Diverse effects of formate on the dissimilatory metabolism of nitrate in Pseudomonas denitrificans ATCC 13867: Growth,nitrite accumulation in culture,cellular activities of nitrate and nitrite reductases

The growth of Pseudomonas denitrificans ATCC 13867 under denitrifying conditions was significantly stimulated by adding an appropriate amount of formate (2.5 mM or above) to the growth medium. The accumulation of nitrite in the culture was markedly depressed so long as formate remained in the culture above a certain level. Cellular activities of enzymes participating in denitrification also changed. The cells grown in the presence of formate exhibited a lower nitrate reductase activity and, in contrast, a higher nitrite reductase activity than the cells grown without added formate.     

Chemical modification studies of tryptophan,arginine and lysine residues in maize chloroplast ferredoxin:sulfite oxidoreductase

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (E _m) values of the siroheme and [4Fe–4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.     

An iron-containing superoxide dismutase from the chemolithotrophic Thiobacillus denitrificans “RT” strain

Superoxide dismutase has been purified to homogeneity from aerobically grown Thiobacillus denitrificans strain RT. It has a molecular weight of 43,000, is composed of two identical subunits which are not covalently bound, and contains 1.35 atom of iron per molecule. Absorption spectra and amino acid analysis are similar to those of other Fe-superoxide dismutases from bacteria. Aerobically and anaerobically grown cells contain the same Fe-enzyme with similar levels of activity. Manometric sulfite oxidation measurements suggest for the enzyme a protective function of sulfite against the autooxidation initiated by superoxide free radicals.Non-Standard Abbreviations DMSO dimethyl sulfoxide - SDS sodium dodecyl sulfate - SOD superoxide dismutase     

biochemical reaction mechanisms in sulfur oxidation by chemosynthetic bacteria

Summary Aspects of the biochemistry of the oxidation of inorganic sulfur compounds are discussed in thiobacilli but chiefly inThiobacillus denitrificans. Almost all of the thiobacilli (e.g. T. denitrificans, T. neapolitanus, T. novellus, andThiobacillus A ₂) were capable of producing approximately 7.5 moles of sulfuric acid aerobically from 3.75 moles of thiosulfate per gram of cellular protein per hr. By far the most prolific producer of sulfuric acid (or sulfates) from the anaerobic thiosulfate oxidation with nitrates wasT. denitrificans which was capable of producing 15 moles of sulfates from 7.5 moles of thiosulfate with concomitant reduction of 12 moles of nitrate resulting in the evolution of 6 moles of nitrogen gas/g protein/hr. The oxidation of sulfide was mediated by the flavo-protein system and cytochromes ofb, c, o, anda-type. This process was sensitive to flavoprotein inhibitors, antimycin A, and cyanide. The aerobic thiosulfate oxidation on the other hand involved cytochromec : O₂ oxidoreductase region of the electron transport chain and was sensitive to cyanide only. The anaerobic oxidation of thiosulfate byT. denitrificans, however, was severely inhibited by the flavoprotein inhibitors because of the splitting of the thiosulfate molecule into the sulfide and sulfite moieties produced by the thiosulfate-reductase. Accumulation of tetrathionate and to a small extent trithionate and pentathionate occurred during anaerobic growth ofT. denitrificans. These polythionates were subsequently oxidized to sulfate with the concomitant reduction of nitrate to N₂. Intact cell suspensions catalyzed the complete oxidation of sulfide, thiosulfate, tetrathionate, and sulfite to sulfate with the stoichiometric reduction of nitrate, nitrite, nitric oxide, and nitrous oxide to nitrogen gas thus indicating that NO₂ ⁻, NO, and N₂O are the possible intermediates in the denitrification of nitrate. This process was mediated by the cytochrome electron transport chain and was sensitive to the electron transfer inhibitors. The oxidation of sulfite involved cytochrome-linked sulfite oxidase as well as the APS-reductase pathways. The latter was absent inT. novellus andThiobacillus A ₂. In all of the thiobacilli the inner as well as the outer sulfur atoms of thiosulfate were oxidized at approximately the same rate by intact cells. The sulfide oxidation occurred in two stages: (a) a cellular-membrane-associated initial and rapid oxidation reaction which was dependent upon sulfide concentration, and (b) a slower oxidation reaction stage catalyzed by the cellfree extracts, probably involving polysulfides. InT. novellus andT. neapolitanus the oxidation of inorganic sulfur compounds is coupled to energy generation through oxidative phosphorylation, however, the reduction of pyridine nucleotides by sulfur compounds involved an energy-linked reversal of electron transfer. Paper read at the Symposium on the Sulphur Cycle, Wageningen, May 1974. Summary already inserted on p. 189 of the present volume.