Sulfite Reduction Research Articles

The Crystal Structure of Desulfovibrio vulgaris Dissimilatory Sulfite Reductase Bound to DsrC Provides Novel Insights into the Mechanism of Sulfate Respiration

Sulfate reduction is one of the earliest types of energy metabolism used by ancestral organisms to sustain life. Despite extensive studies, many questions remain about the way respiratory sulfate reduction is associated with energy conservation. A crucial enzyme in this process is the dissimilatory sulfite reductase (dSiR), which contains a unique siroheme-[4Fe4S] coupled cofactor. Here, we report the structure of desulfoviridin from Desulfovibrio vulgaris, in which the dSiR DsrAB (sulfite reductase) subunits are bound to the DsrC protein. The alpha(2)beta(2)gamma(2) assembly contains two siroheme-[4Fe4S] cofactors bound by DsrB, two sirohydrochlorins and two [4Fe4S] centers bound by DsrA, and another four [4Fe4S] centers in the ferredoxin domains. A sulfite molecule, coordinating the siroheme, is found at the active site. The DsrC protein is bound in a cleft between DsrA and DsrB with its conserved C-terminal cysteine reaching the distal side of the siroheme. We propose a novel mechanism for the process of sulfite reduction involving DsrAB, DsrC, and the DsrMKJOP membrane complex (a membrane complex with putative disulfide/thiol reductase activity), in which two of the six electrons for reduction of sulfite derive from the membrane quinone pool. These results show that DsrC is involved in sulfite reduction, which changes the mechanism of sulfate respiration. This has important implications for models used to date ancient sulfur metabolism based on sulfur isotope fractionations.

Electrochemical reduction of SO2 on poly-Co-3-tetraaminophenylporphyrin glassy carbon modified electrode¶

This article describes the electrochemical reduction of sulfite in water–ethanol mixtures under acidic conditions on a glassy carbon electrode modified with a conducting film formed by Co(II) tetra-3-aminophenyl porphyrin. Rotating disc electrode experiments show a kinetic limitation related with the formation of an adduct between the reduced porphyrin and the sulfur compound. UV–Visible spectroelectrochemical data suggest that Co(I) is not stabilized; thus the adduct should involve a radical species of the porphyrin. However, the resulting modified electrode is stable and shows a linear relationship between current and concentration of the sulfur species at pH 1. The detection range of sulfite by the modified electrode goes from 12 to 150 mg L−1. The detection limit is 4.15 mg L−1. ¶This article is dedicated to Professor A. Mederos.

Purification, crystallization and preliminary crystallographic analysis of a dissimilatory DsrAB sulfite reductase in complex with DsrC

Dissimilatory sulfite reductase (dSiR, DsrAB) is a key protein in dissimilatory sulfur metabolism, one of the earliest types of energy metabolism to be traced on earth. dSirs are large oligomeric proteins around 200kDa forming an alpha(2)beta(2) arrangement and including a unique siroheme-[4Fe-4S] coupled cofactor. Here, we report the purification, crystallization and preliminary X-ray diffraction analysis of dSir isolated from Desulfovibrio vulgaris Hildenborough, also known as desulfoviridin. In this enzyme the DsrAB protein is associated with DsrC, a protein of unknown function that is believed to play an important role in the sulfite reduction. Crystals belong to the monoclinic space group P2(1) with unit-cell parameters a=122.7, b=119.4 and c=146.7A and beta =110.0 degrees , and diffract X-rays to 2.8A on a synchrotron source.

Structure of the Dissimilatory Sulfite Reductase from the Hyperthermophilic Archaeon Archaeoglobus fulgidus

Conservation of energy based on the reduction of sulfate is of fundamental importance for the biogeochemical sulfur cycle. A key enzyme of this ancient anaerobic process is the dissimilatory sulfite reductase (dSir), which catalyzes the six-electron reduction of sulfite to hydrogen sulfide under participation of a unique magnetically coupled siroheme–[4Fe–4S] center. We determined the crystal structure of the enzyme from the sulfate-reducing archaeon Archaeoglobus fulgidus at 2-Å resolution and compared it with that of the phylogenetically related assimilatory Sir (aSir). dSir is organized as a heterotetrameric (αβ) 2 complex composed of two catalytically independent αβ heterodimers. In contrast, aSir is a monomeric protein built of two fused modules that are structurally related to subunits α and β except for a ferredoxin domain inserted only into the subunits of dSir. The [4Fe–4S] cluster of this ferredoxin domain is considered as the terminal redox site of the electron transfer pathway to the siroheme–[4Fe–4S] center in dSir. While aSir binds one siroheme–[4Fe–4S] center, dSir harbors two of them within each αβ heterodimer. Surprisingly, only one siroheme–[4Fe–4S] center in each αβ heterodimer is catalytically active, whereas access to the second one is blocked by a tryptophan residue. The spatial proximity of the functional and structural siroheme–[4Fe–4S] centers suggests that the catalytic activity at one active site was optimized during evolution at the expense of the enzymatic competence of the other. The sulfite binding mode and presumably the mechanism of sulfite reduction appear to be largely conserved between dSir and aSir. In addition, a scenario for the evolution of Sirs is proposed.

Isolation and characterization of an operon involved in sulfate and sulfite metabolism inSinorhizobium fredii

A gene cluster ORFabcd from a Sinorhizobium fredii HN01 mutant strain HSMRalpha was isolated. We showed that it was an operon involved in sulfur metabolism. Functional studies revealed that, except for ORFb, the three genes ORFa, ORFc and ORFd were involved in sulfite reduction. ORFa and ORFc were similar to the cysG and cysI from Sinorhizobium meliloti 1021 and Rhizobium etli CFN 42, respectively. ORFd encodes a conserved hypothetical protein in other bacteria. We demonstrate here, for the first time, that it was a new locus involved in sulfate assimilation in S. fredii HN01 and we designated it as cysII.

The conservation of iron objects in archaeological preservation – Application and further development of alkaline sulphite method for conservation of large quantities of iron finds

AbstractThe usual damage on archaeological iron finds caused by corrosion are outlined in this paper. For a better understanding of these phenomena and the resulting consequences for iron conservation a short literature summary is given regarding actual corrosion models for archaeological iron finds. The desalination of chloride‐contaminated iron finds is the demand to be derived from this synopsis. Therefore, the desalination with the alkaline sulphite reduction method is applied at Landesamt für Denkmalpflege Baden‐Württemberg recently, which allows the long‐term stabilization of many iron finds. Finally, the research project ‘Investigations into the alkaline sulphite treatment for conservation of large quantities of archaeological iron objects’ is introduced.

Dinaphthotetraaza[14]annulene copper(II) complexes in the electrocatalytic reduction of carbon dioxide and bisulfite anion

A modified synthetic route for the complexes [Cu(II)5,7,12,14-tetramethyldinaphtho [b,i][1,4,8,11]tetraaza[14]annulene], [Cu(II)tmdnTAA], and [Cu(II) 5,7,12,14-tetramethyl-6,13-dichloro-dinaphtho[b,i][1,4,8,11]tetraaza[14]annulene], [Cu(II)dCltmdnTAA], is presented in this work. The electrochemical characterization of both complexes and their precursors, [bis(2,4-pentanedionato)copper(II)], [Cu(II)(acac)2] and [bis(3-chloro-2,4-pentanedionato)copper(II)], [Cu(II)(3-Cl-acac)2], respectively, under nitrogen and carbon dioxide is also presented. The voltammetric response of [Cu(II)(acac)2] and [Cu(II)(3-Cl-acac)2] are different compared to [Cu(II)tmdnTAA] and [Cu(II)dCltmdnTAA] under nitrogen. Precursors show the reduction of Cu(I) to Cu(0) and the tetraazadinaphtho[14]annulene complexes do not. The chlorine substituted complex has a lower reduction potential than the unsubstituted homologue under nitrogen atmosphere. However, the contrary response is obtained in the presence of carbon dioxide: the unsubstituted complex is more catalytic in terms of potential because the current discharge appears 270 mV shifted to the anodic region. These facts can be explained in terms of electronic and steric effects. The modified electrode obtained by oxidative electropolymerization of [Cu(II)tmdnTAA] over glassy carbon electrode presented a suitable amperometric response for the sulfite reduction in acidic medium (pH = 2.7). A linear correlation was observed for the catalytic current and sulfite concentration between 0.6–6.0 mM range.

Binding and Reduction of Sulfite by Cytochrome c Nitrite Reductase,

Pentaheme cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron reduction of nitrite to ammonia as the final step in the dissimilatory pathway of nitrate ammonification. It has also been shown to reduce sulfite to sulfide, thus forming the only known link between the biogeochemical cycles of nitrogen and of sulfur. We have found the sulfite reductase activity of ccNiR from Wolinella succinogenes to be significantly smaller than its nitrite reductase activity but still several times higher than the one described for dissimilatory, siroheme-containing sulfite reductases. To compare the sulfite reductase activity of ccNiR with our previous data on nitrite reduction, we determined the binding mode of sulfite to the catalytic heme center of ccNiR from W. succinogenes at a resolution of 1.7 A. Sulfite and nitrite both provide a pair of electrons to form the coordinative bond to the Fe(III) active site of the enzyme, and the oxygen atoms of sulfite are found to interact with the three active site protein residues conserved within the enzyme family. Furthermore, we have characterized the active site variant Y218F of ccNiR that exhibited an almost complete loss of nitrite reductase activity, while sulfite reduction remained unaffected. These data provide a first direct insight into the role of the first sphere of protein ligands at the active site in ccNiR catalysis.

Biosynthesis of Cysteine.

The synthesis of L-cysteine from inorganic sulfur is the predominant mechanism by which reduced sulfur is incorporated into organic compounds. L-cysteineis used for protein and glutathione synthesis and serves as the primary source of reduced sulfur in L-methionine, lipoic acid, thiamin, coenzyme A (CoA), molybdopterin, and other organic molecules. Sulfate and thiosulfate uptake in E. coli and serovar Typhimurium are achieved through a single periplasmic transport system that utilizes two different but similar periplasmic binding proteins. Kinetic studies indicate that selenate and selenite share a single transporter with sulfate, but molybdate also has a separate transport system. During aerobic growth, the reduction of sulfite to sulfide is catalyzed by NADPH-sulfite reductase (SiR), and serovar Typhimurium mutants lacking this enzyme accumulate sulfite from sulfate, implying that sulfite is a normal intermediate in assimilatory sulfate reduction. L-Cysteine biosynthesis in serovar Typhimurium and E. coli ceases almost entirely when cells are grown on L-cysteine or L-cystine, owing to a combination of end product inhibition of serine transacetylase by L-cysteine and a gene regulatory system known as the cysteine regulon, wherein genes for sulfate assimilation and alkanesulfonate utilization are expressed only when sulfur is limiting. In vitro studies with the cysJIH, cysK, and cysP promoters have confirmed that they are inefficient at forming transcription initiation complexes without CysB and N-acetyl-L-serine. Activation of the tauA and ssuE promoters requires Cbl. It has been proposed that the three serovar Typhimurium anaerobic reductases for sulfite, thiosulfate, and tetrathionate may function primarily in anaerobic respiration.

Treatment of industrial wastewater using photooxidation and bioaugmentation technology

The versatile metabolism of microorganisms has an played important role in the biodegradation of recalcitrant toxic compounds entering into the natural environment. The biodegradability of organics can be enhanced using bioaugmentation and advanced oxidation processes (AOP) for aerobic/anaerobic treatment programs. Wastewater from a bulk drug (cresol) plant had high levels of TDS, COD and BOD, whilst the levels from a pigment plant low. Both contained organics difficult to degrade. AOP using hydroxyl radical generated in 1 L glass reactor using UV and H2O2 efficiently oxidised phenol and cresol. COD and sulphite reduction in cresol containing wastewater were 20-60% in 1-6 h. A twenty to thirty percent reduction in copper phthalocyanine pigment effluents was achieved in 6 h using AOP. Strains of Micrococcus, Pseudomonas, and Nocardia degrading phenol, cresol were isolated from soil and sludge. Mixed biomass of these organisms removed phenols (1,000 ppm) and cresols (500 ppm) completely in 24 and 72 h, respectively. The COD and BOD reductions under the optimum nutritional and physiological conditions were in the range of 70 to 90%. When added to the bioreactor, 20% of the developed biomass of mixed strains of Micrococcus, Nocardia and Pseudomonas increased the rate of COD and BOD reduction gradually and stabilised at 80-90%. Added biomass improved the overall efficiency of the aerobic process.

Sulfite Reduction in Mycobacteria

Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [k(cat) = 1.0 (+/-0.1) electron consumed per second, and K(m(SO(3)(-2))) = 27 (+/-1) microM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe(2+) into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery.

Niches of the pre‐photosynthetic biosphere and geologic preservation of Earth's earliest ecology

ABSTRACTThe tree of terrestrial life probably roots in non‐photosynthetic microbes. Chemoautotrophs were the first primary producers, and the globally dominant niches in terms of primary productivity were determined by availability of carbon dioxide and hydrogen for methanogenesis and sulfite reduction. Methanogen niches were most abundant where CO2‐rich ocean water flowed through serpentinite. Black smoker vents from basalt supplied comparable amount of H2. Hydrogen from arc volcanoes supported a significant methanogenic niche at the Earth's surface. SO2from arc volcanoes reacted with organic matter and hydrogen, providing a significant surface niche. Methane ascended to the upper atmosphere where photolysis produced C‐rich haze and CO, and H escaped into space. The CO and C‐rich haze supported secondary surface niches. None of these ecologies were bountiful; less than 1% of the CO2vented by ridge axes, arcs, and metamorphism became organic matter before it was buried in carbonate. In contrast, a photosynthetic biosphere leaves copious amounts of organic carbon, locally concentrated in sediments. Black shales are a classic geologic biosignature for photosynthesis that can survive subduction and high‐grade metamorphism.

DNA binding and partial nucleoid localization of the chloroplast stromal enzyme ferredoxin:sulfite reductase.

Sulfite reductase (SiR) is an important enzyme catalyzing the reduction of sulfite to sulfide during sulfur assimilation in plants. This enzyme is localized in plastids, including chloroplasts, and uses ferredoxin as an electron donor. Ferredoxin-dependent SiR has been found in isolated chloroplast nucleoids, but its localization in vivo or in intact plastids has not been examined. Here, we report the DNA-binding properties of SiRs from pea (PsSiR) and maize (ZmSiR) using an enzymatically active holoenzyme with prosthetic groups. PsSiR binds to both double-stranded and single-stranded DNA without significant sequence specificity. DNA binding did not affect the enzymatic activity of PsSiR, suggesting that ferredoxin and sulfite are accessible to SiR molecules within the nucleoids. Comparison of PsSiR and ZmSiR suggests that ZmSiR does indeed have DNA-binding activity, as was reported previously, but the DNA affinity and DNA-compacting ability are higher in PsSiR than in ZmSiR. The tight compaction of nucleoids by PsSiR led to severe repression of transcription activity in pea nucleoids. Indirect immunofluorescence microscopy showed that the majority of SiR molecules colocalized with nucleoids in pea chloroplasts, whereas no particular localization to nucleoids was detected in maize chloroplasts. These results suggest that SiR plays an essential role in compacting nucleoids in plastids, but that the extent of association of SiR with nucleoids varies among plant species.

Reactions of the amine-containing drugs fluoxetine and metoprolol during chlorination and dechlorination processes used in wastewater treatment

The reactivities of the amine-containing pharmaceuticals fluoxetine and metoprolol with hypochlorite were studied using conditions that simulate wastewater disinfection including neutral pH (7.0), a range of reaction times (2–60 min), and a molar excess of hypochlorite relative to the pharmaceutical concentration (5.7 times). The reactions were monitored using liquid chromatography (LC) with several detection modes including ultraviolet absorbance (UV), mass spectrometry (MS), and post-column reaction/reductive electrochemistry (EC) for determining active chlorine products. At levels of 10 μM, both compounds reacted rapidly (<2 min) to form principally N-chloramine products that were stable in aqueous solution for at least 1 h. The reaction was also studied in wastewater, and similar reactivity was noted. These results demonstrate that the cations fluoxetine and metoprolol are likely to be rapidly transformed into neutral N-chloramines during wastewater disinfection. The reactivity of the N-chloramines was also studied with sulfite to simulate dechlorination, which is often employed in wastewater treatment. Both N-chloramines reacted slowly with sulfite. In the pure water dechlorination experiments, it was estimated that 70% and 10% of the peak areas remained after 2 min reaction time for fluoxetine and metoprolol, respectively. At longer reaction times both N-chloramines had been completely reduced by sulfite, and the product of the sulfite reduction reaction was the parent pharmaceutical amine. Since typical dechlorination times in wastewater treatment are on the order of seconds, this suggests the chloramines formed from these two basic drugs might evade dechlorination and be released into the environment. The implications of chloramine release are discussed.

Multielectronic redox and electrocatalytic supramolecular films based on a tetraruthenated iron porphyrin

Layer-by-layer electrostatic assembled Fe-3TRPyP/CuTSPc supramolecular films constituted by homogeneously dispersed electrochemically active columnar structures, about 15nm high, were obtained and characterized. They are redox conductors acting as rectifiers in the presence of redox species in solution, providing an excellent electrocatalyst for both oxidation and reduction of nitrite and sulfite. The spectroelectrochemical characterization of the supermolecular porphyrin in solution (−2.6 to 2.3V) revealed 11 redox processes involving up to 26 electrons, allowing further applications of the thin films as multi electron-transfer catalysts and active materials for molecular devices.

Electrocatalysis on tetraruthenated nickel and cobalt porphyrins electrostatic assembled films

Abstract The meta and para-pyridine substituted isomers of nickel and cobalt meso-pyridylporphyrins coordinated to four [Ru(bpy)2Cl]+ complexes, M(TRPyP), have been obtained and fully investigated by means of electrochemistry and spectroelectrochemistry. These compounds form stable layer-by-layer electrostatic assembled films with tetrasulfonated phthalocyaninate cuprate(II) anion, exhibiting distinct electrocatalytic activity for sulfite and nitrite oxidation and reduction reactions. Interestingly, M(3-TRPyP)/CuTSPc films showed good electrocatalytic activity for the oxidation and reduction of sulfite and nitrite at pH 4.7, while at pH 6.8 both M(TRPyP) derivatives were active only for the oxidation process. Such differences were attributed to electronic structure of MTRPyPs and changes in the active site environment caused by the distinct structure and packing properties of the meta- and para-isomers, rather than to differences in the redox potentials. Summarizing, supramolecular electrocatalytic activity was demonstrated for those electrostatic assembled films introducing a new paradigm for the development of chemical sensors for simultaneous analysis of multiple substrates.

Effect of competitive terminal electron acceptor processes on dechlorination of cis-1,2-dichloroethene and 1,2-dichloroethane in constructed wetland soils

Thermodynamic calculations were coupled with time-series measurements of chemical species (parent and daughter chlorinated solvents, H(2), sulfite, sulfate and methane) to predict the anaerobic transformation of cis-1,2-dichloroethene (cis-1,2-DCE) and 1,2-dichloroethane (1,2-DCA) in constructed wetland soil microcosms inoculated with a dehalorespiring culture. For cis-1,2-DCE, dechlorination occurred simultaneously with sulfite and sulfate reduction but competitive exclusion of methanogenesis was observed due to the rapid H(2) drawdown by the dehalorespiring bacteria. Rates of cis-1,2-DCE dechlorination decreased proportionally to the free energy yield of the competing electron acceptor and proportionally to the rate of H(2) drawdown, suggesting that H(2) competition between dehalorespirers and other populations was occurring, affecting the dechlorination rate. For 1,2-DCA, dechlorination occurred simultaneously with methanogenesis and sulfate reduction but occurred only after sulfite was completely depleted. Rates of 1,2-DCA dechlorination were unaffected by the presence of competing electron-accepting processes. The absence of a low H(2) threshold suggests that 1,2-DCA dechlorination is a cometabolic transformation, occurring at a higher H(2) threshold, despite the high free energy yields available for dehalorespiration of 1,2-DCA. We demonstrate the utility of kinetic and thermodynamic calculations to understand the complex, H(2)-utilizing reactions occurring in the wetland bed and their effect on rates of dechlorination of priority pollutants.

NMR Study of the Electron Transfer Complex of Plant Ferredoxin and Sulfite Reductase: MAPPING THE INTERACTION SITES OF FERREDOXIN

Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP(+) reductase is the interaction partner.

Evaluation of acid phosphatase as a confirmation test for Clostridium perfringens isolated from water

To evaluate testing for acid phosphatase as an alternative method for the confirmation of Clostridium perfringens isolated from water. Sixty-two reference strains of Clostridium were tested for their ability to produce acid phosphatase, as well as reduction of sulfite on tryptose sulfite cycloserine agar (TSC) and production of fluorescence in TSC supplemented with 4-methylumbelliferylphosphate (MUP). Additionally 155 environmental presumptive C. perfringens isolates from TSC incubated at 44 degrees C were identified and tested for acid phosphatase production and by the conventional MNLG (testing for motility, nitrate reduction, lactose fermentation and gelatin liquefaction) confirmation procedure. Twenty-seven strains from 15 species of Clostridium-reduced sulfite to some extent on TSC incubated at 44 degrees C, with a significant number of species being able to grow well at this temperature, indicating that a confirmation step is needed for the enumeration of C. perfringens on this medium. All 10 strains of C. perfringens tested, together with one strain each of Clostridium baratii and Clostridium rectum produced acid phosphatase. These also produced fluorescence on MUP supplemented TSC, as did 13 strains of acid phosphatase negative, sulfite-reducing clostridia, representing nine species. Of the environmental isolates, 114 were identified as C. perfringens of which 108 (94.7%) were confirmed by the acid phosphatase test compared with 104 (91.2%) by the MNLG tests. Testing for acid phosphatase production is at least as reliable, and much simpler to perform, than the current standard confirmation MNLG procedure. Incorporation of MUP into TSC does not reliably improve the identification of presumptive C. perfringens. Application of testing for acid phosphatase as a confirmation test for C. perfringens would substantially simplify the analysis for this bacterium from water samples, and reduce the analysis time to confirmed counts.

Molecular and catalytic properties of a novel cytochrome c nitrite reductase from nitrate-reducing haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens

A highly active cytochrome c nitrite reductase from the haloalkaliphilic sulfur-oxidizing non-ammonifying bacterium Tv. nitratireducens strain ALEN 2 (TvNiR) was isolated and purified to apparent electrophoretic homogeneity. The enzyme catalyzes reductive conversion of nitrite and hydroxylamine to ammonia without release of any intermediates, as well as reduction of sulfite to sulfide. TvNiR also possesses peroxidase activity. In solution TvNiR exists as a stable hexamer with molecular mass of about 360 kDa. Each TvNiR subunit with molecular mass of 64 kDa contains, as defined from spectral properties and sequence analysis, eight c-type haems. Seven of them are coordinated by the characteristic CXXCH motifs for haem c binding, while one is bonded by the unique CXXCK motif. So far, this motif coordinating the catalytic haem was found only in bacterial cytochrome c nitrite reductases (ccNiRs). All the residues essential for catalysis in the known ccNiRs were also identified in TvNiR. However, TvNiR is only distantly related to known bacterial ammonifying dissimilatory ccNiRs, sharing no more than 20% homology.